PD1 and/or LAG3 binders

ABSTRACT

The present invention provides molecules, such as ISVDs and Nanobodies, that bind to PD1 and LAG3 and, optionally to human serum albumin. These molecules have been engineered so as to reduce the incidence of binding by pre-existing antibodies in the bodies of a subject administered such a molecule. Methods for increasing immune response, treating cancer and/or treating an infectious disease with such molecules are provided.

This Application claim the benefit of U.S. Provisional PatentApplication No. 62/257,009, filed Nov. 18, 2015; which is hereinincorporated by referenced in its entirety

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “24238_US_NP_SEQTXT_01OCTOBER2018.txt”, creation date of Oct.1, 2018, and a size of 203 Kb. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates, in part, to amino acid sequences bindingto programmed cell death protein 1 (“PD1”), e.g., human PD1 andlymphocyte activation gene 3 (LAG3). In particular, the presentinvention relates, in part, to improved heavy-chain immunoglobulinsingle variable domains (also referred to herein as “ISVs” or “ISVDs”)binding to PD1 and LAG3, as well as to polypeptides and other compoundsthat comprise such ISVDs. Other aspects, embodiments, features, uses andadvantages of the invention will be clear to the skilled person based onthe disclosure herein.

BACKGROUND OF THE INVENTION

Programmed death receptor 1 (PD-1) is an immunoinhibitory receptor thatis primarily expressed on activated T and B cells. Interaction with itsligands has been shown to attenuate T-cell responses both in vitro andin vivo. Blockade of the interaction between PD-1 and one of itsligands, PD-L1, has been shown to enhance tumor-specific CD8⁺ T-cellimmunity and may therefore be helpful in clearance of tumor cells by theimmune system.

PD-1 (encoded by the gene Pdcd1) is an Immunoglobulin superfamily memberrelated to CD28, and CTLA-4. PD-1 has been shown to negatively regulateantigen receptor signaling upon engagement of its ligands (PD-L1 and/orPD-L2) The structure of murine PD-1 has been solved as well as theco-crystal structure of mouse PD-1 with human PD-L1 (Zhang, X. et al.,Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105:3011-6 (2008)). PD-1 and like family members are type I transmembraneglycoproteins containing an Ig Variable-type (V-type) domain responsiblefor ligand binding and a cytoplasmic tail that is responsible for thebinding of signaling molecules. The cytoplasmic tail of PD-1 containstwo tyrosine-based signaling motifs, an ITIM (immunoreceptortyrosine-based inhibition motif) and an ITSM (immunoreceptortyrosine-based switch motif).

Following T cell stimulation, PD-1 recruits the tyrosine phosphataseSHP-2 to the ITSM motif within its cytoplasmic tail, leading to thedephosphorylation of effector molecules such as CD3 zeta, PKC theta andZAP70 that are involved in the CD3 T cell signaling cascade. Themechanism by which PD-1 downmodulates T cell responses is similar to,but distinct from that of CTLA-4, as both molecules regulate anoverlapping set of signaling proteins (Parry et al., Mol. Cell. Biol.25: 9543-9553 (2005)). Bennett and coworkers have shown thatPD-1-mediated inhibition of T-cell signaling is only effective when bothactivating and inhibitory signals are on the same surface, indicatingthat the PD-1 signaling mechanism is spatiotemporally determined(Bennett F. et al., J Immunol. 170:711-8 (2003)). PD-1 was shown to beexpressed on activated lymphocytes (peripheral CD4⁺ and CD8⁺ T cells, Bcells and monocytes) and has also been shown to be expressed duringthymic development on CD4⁻CD8⁻ (double negative) T cells as well as NK-Tcells.

The ligands for PD-1 (PD-L1 and PD-L2) are constitutively expressed orcan be induced in a variety of cell types, including non-hematopoietictissues as well as various tumor types. PD-L1 is expressed on B, T,myeloid and dendritic cells (DCs), but also on peripheral cells, likemicrovascular endothelial cells and non-lymphoid organs like heart, lungetc. In contrast, PD-L2 is only found on macrophages and DCs. Theexpression pattern of PD-1 ligands is suggestive of a role for PD-1 inmaintaining peripheral tolerance and may serve to regulate self-reactiveT- and B-cell responses in the periphery. Both ligands are type Itransmembrane receptors containing both IgV- and IgC-like domains in theextracellular region. Both ligands contain short cytoplasmic regionswith no known signaling motifs.

Interaction of PD-1 with its ligands leads to the inhibition oflymphocyte proliferation in vitro and in vivo. Disruption of thePD-1/PD-L1 interaction has been shown to increase T cell proliferationand cytokine production and block progression of the cell cycle. Initialanalysis of Pdcd1^(−/−) mice did not identify any drastic immunologicalphenotype. However aged mice developed spontaneous autoimmune diseaseswhich differ according to the strain onto which the Pdcdl deficiency wasbackcrossed. These include lupus-like proliferative arthritis (C57BL/6)(Nishimura H. et al., Int. Immunol. 10: 1563-1572 (1998)), fatalcardiomyopathy (BALB/c) (Nishimura H. et al., Science 291: 319-322(2001)) and type I diabetes (NOD) (Wang J. et al., Proc. Natl. Acad.Sci. USA 102: 11823-11828 (2005)). Overall, analysis of the knockoutanimals has led to the understanding that PD-1 functions mainly ininducing and regulating peripheral tolerance. Thus, therapeutic blockadeof the PD-1 pathway may be helpful in overcoming immune tolerance. Suchselective blockade may be of use in the treatment of cancer or infectionas well as in boosting immunity during vaccination (either prophylacticor therapeutic).

The role of PD-1 in cancer is established in the literature. It is knownthat tumor microenvironment can protect tumor cells from efficientimmune destruction. PD-L1 has recently been shown to be expressed on anumber of mouse and human tumors (and is inducible by IFN gamma on themajority of PD-L1 negative tumor cell lines) and is postulated tomediate immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A.99: 12293-12297 (2002); Strome S. E. et al., Cancer Res., 63: 6501-6505(2003).

In humans, expression of PD-1 (on tumor infiltrating lymphocytes) and/orPD-L1 (on tumor cells) has been found in a number of primary tumorbiopsies assessed by immunohistochemistry. Such tissues include cancersof the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast,kidney, esophagus, stomach, oral squamous cell, urothelial cell, andpancreas as well as tumors of the head and neck (Brown J. A. et al., J.Immunol. 170: 1257-1266 (2003); Dong H. et al., Nat. Med. 8: 793-800(2002); Wintterle et al., Cancer Res. 63: 7462-7467 (2003); Strome S. E.et al., Cancer Res., 63: 6501-6505 (2003); Thompson R. H. et al., CancerRes. 66: 3381-5 (2006); Thompson et al., Clin. Cancer Res. 13: 1757-61(2007); Nomi T. et al., Clin. Cancer Res. 13: 2151-7. (2007)). Morestrikingly, PD-ligand expression on tumor cells has been correlated topoor prognosis of cancer patients across multiple tumor types (reviewedin Okazaki and Honjo, Int. Immunol. 19: 813-824 (2007)).

Blockade of the PD-1/PD-L1 interaction could lead to enhancedtumor-specific T-cell immunity and therefore be helpful in clearance oftumor cells by the immune system. To address this issue, a number ofstudies were performed. In a murine model of aggressive pancreaticcancer, T. Nomi et al. (Clin. Cancer Res. 13: 2151-2157 (2007))demonstrated the therapeutic efficacy of PD-1/PD-L1 blockade.Administration of either PD-1 or PD-L1 directed antibody significantlyinhibited tumor growth. Antibody blockade effectively promoted tumorreactive CD8⁺ T cell infiltration into the tumor resulting in theup-regulation of anti-tumor effectors including IFN gamma, granzyme Band perforin. Additionally, the authors showed that PD-1 blockade can beeffectively combined with chemotherapy to yield a synergistic effect. Inanother study, using a model of squamous cell carcinoma in mice,antibody blockade of PD-1 or PD-L1 significantly inhibited tumor growth(Tsushima F. et al., Oral Oncol. 42: 268-274 (2006)).

In other studies, transfection of a murine mastocytoma line with PD-L1led to decreased lysis of the tumor cells when co-cultured with atumor-specific CTL clone. Lysis was restored when anti-PD-L1 mAb wasadded (Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99: 12293-12297(2002)). In vivo, blocking the PD1/PD-L1 interaction was shown toincrease the efficacy of adoptive T cell transfer therapy in a mousetumor model (Strome S. E. et al., Cancer Res. 63: 6501-6505 (2003)).Further evidence for the role of PD-1 in cancer treatment comes fromexperiments performed with PD-1 knockout mice. PD-L1 expressing myelomacells grew only in wild-type animals (resulting in tumor growth andassociated animal death), but not in PD-1 deficient mice (Iwai Y. etal., Proc. Natl. Acad. Sci. U.S.A. 99: 12293-12297 (2002)).

In human studies, R. M. Wong et al. (Int. Immunol. 19: 1223-1234 (2007))showed that PD-1 blockade using a fully human anti-PD-1 antibodyaugmented the absolute numbers of tumor-specific CD8+ T cells (CTLs) inex vivo stimulation assays using vaccine antigens and cells fromvaccinated individuals. In a similar study, antibody blockade of PD-L1resulted in enhanced cytolytic activity of tumor-associatedantigen-specific cytotoxic T cells and increased cytokine production bytumor specific T_(H) cells (Blank C. et al., Int. J. Cancer 119: 317-327(2006)). The same authors showed that PD-L1 blockade augmentstumor-specific T cell responses in vitro when used in combination withanti-CTLA-4 blockade.

Overall, the PD-1/PD-L1 pathway is a well-validated target for thedevelopment of antibody therapeutics for cancer treatment. Anti-PD-1antibodies may also be useful in chronic viral infection. Memory CD8⁺ Tcells generated after an acute viral infection are highly functional andconstitute an important component of protective immunity. In contrast,chronic infections are often characterized by varying degrees offunctional impairment (exhaustion) of virus-specific T-cell responses,and this defect is a principal reason for the inability of the host toeliminate the persisting pathogen. Although functional effector T cellsare initially generated during the early stages of infection, theygradually lose function during the course of a chronic infection. Barberet al. (Barber et al., Nature 439: 682-687 (2006)) showed that miceinfected with a laboratory strain of LCMV developed chronic infectionresulting in high levels of virus in the blood and other tissues. Thesemice initially developed a robust T cell response, but eventuallysuccumbed to the infection upon T cell exhaustion. The authors foundthat the decline in number and function of the effector T cells inchronically infected mice could be reversed by injecting an antibodythat blocked the interaction between PD-1 and PD-L1.

Recently, it has been shown that PD-1 is highly expressed on T cellsfrom HIV infected individuals and that receptor expression correlateswith impaired T cell function and disease progression (Day et al.,Nature 443:350-4 (2006); Trautmann L. et al., Nat. Med. 12: 1198-202(2006)). In both studies, blockade of the ligand PD-L1 significantlyincreased the expansion of HIV-specific, IFN-gamma producing cells invitro.

Other studies also implicate the importance of the PD-1 pathway incontrolling viral infection. PD-1 knockout mice exhibit better controlof adenovirus infection than wild-type mice (Iwai et al., J. Exp. Med.198:39-50 (2003)). Also, adoptive transfer of HBV-specific T cells intoHBV transgenic animals initiated hepatitis (Isogawa M. et al., Immunity23:53-63 (2005)). The disease state of these animals oscillates as aconsequence of antigen recognition in the liver and PD-1 upregulation byliver cells.

In addition, LAG3 (CD223) is a cell surface molecule expressed onactivated T cells (Huard et al. Immunogenetics 39:213-217, 1994), NKcells (Triebel et al. J Exp Med 171:1393-1405, 1990), B cells (Kisielowet al. Eur J Immunol 35:2081-2088, 2005), and plasmacytoid dendriticcells (Workman et al. J Immunol 182:1885-1891, 2009) that plays animportant role in the function of these lymphocyte subsets. In addition,the interaction between LAG3 and its major ligand, Class II MHC, isthought to play a role in modulating dendritic cell function (Andreae etal. J Immunol 168:3874-3880, 2002). Recent preclinical studies havedocumented a role for LAG-3 in CD8 T-cell exhaustion (Blackburn et al.Nat Immunol 10:29-37, 2009).

As with chronic viral infection, tumor antigen-specific CD4⁺ and CD8⁺ Tcells display impaired effector function and an exhausted phenotypecharacterized by decreased production of pro-inflammatory cytokines andhyporesponsiveness to antigenic re-stimulation. This is mediated by cellextrinsic mechanisms, such as regulatory T-cells (Treg), and cellintrinsic mechanisms, such as inhibitory molecules that are upregulatedon exhausted, tumor-infiltrating lymphocytes (TIL). These inhibitorymechanisms represent a formidable barrier to effective antitumorimmunity.

LAG-is expressed on tolerized TILs suggesting that they contribute totumor-mediated immune suppression. Inhibition of LAG3 may lead toenhanced activation of antigen-specific T cells from which a therapeuticbenefit may be gained.

SUMMARY OF THE INVENTION

The present invention encompasses a PD1 binder (e.g., an immunoglobulinsingle variable domain (ISVD) or a Nanobody that binds to PD1 (e.g.,human PD1) comprising the amino acid sequence set forth in SEQ ID NO: 1or 2; but which comprises a mutation at one or more of positions 1, 11,14, 52a, 73, 74, 83, 89, 100a, 110 and 112 wherein said positions arenumbered according to Kabat (e.g., L11V, A14P, A74S, K83R, I89L and,optionally, E1D; or L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100a and,optionally, E1D), comprising CDR1 that comprises the amino acidsequence: IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); CDR2 thatcomprises the amino acid sequence: VITXSGGITYYADSVKG (SEQ ID NO: 4;wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V);and CDR3 that comprises the amino acid sequence: DKHQSSXYDY (SEQ ID NO:5, wherein X is W or F) or DKHQSSXYDY (SEQ ID NO: 8, wherein X is W orF). In an embodiment of the invention, the PD1 binder (e.g., an ISVDsuch as a Nanobody) comprises an amino acid residue at position 11 thatis chosen from L or V; an amino acid residue at position 89 chosen fromT, V or L; an amino acid residue at position 110 chosen from T, K or Q;and/or an amino acid residue at position 112 chosen from S, K or Q. Inan embodiment of the invention, the PD1 binder (e.g., an ISVD such as aNanobody) comprises one or more mutations described is a member selectedfrom the group consisting of: 89T; 89L in combination with 11V; 89L incombination with 110K or 110Q; 89L in combination with 112K or 112Q; 89Lin combination with 11V and 110K or 110Q; 89L in combination with 11Vand 112K or 112Q; 11V in combination with 110K or 110Q; 11V incombination with 112K or 112Q. In an embodiment of the invention, theamino acid at positions 11, 89, 110 and 112 are as any of those setforth in the Table B herein. In an embodiment of the invention, the PD1binder (e.g., an ISVD such as a Nanobody) comprises one or moremutations at a position selected from the group consisting of 1, 14, 41,74, 83, 87 and 108 and/or one or more humanizing substitutions known perse (for which reference is made to the prior art cited herein, such asWO 08/020079 or WO 09/138519. In an embodiment of the invention, the PD1binder (e.g., an ISVD such as a Nanobody) comprises a C-terminalextension of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. For example,in an embodiment of the invention, the PD1 binder (e.g., an ISVD such asa Nanobody) comprises a C-terminal extension according to the formula—X(n), wherein X and n are as follows: (a) n=1 and X=Ala; (b) n=2 andeach X=Ala; (c) n=3 and each X=Ala; (d) n=2 and at least one X=Ala andwherein the remaining amino acid residue(s) X are independently chosenfrom any naturally occurring amino acid; (e) n=3 and at least one X=Alaand wherein the remaining amino acid residue(s) X are independentlychosen from any naturally occurring amino acid; (f) n=3 and at least twoX=Ala and wherein the remaining amino acid residue(s) X areindependently chosen from any naturally occurring amino acid; (g) n=1and X=Gly; (h) n=2 and each X=Gly; (i) n=3 and each X=Gly; (j) n=2 andat least one X=Gly wherein the remaining amino acid residue(s) X areindependently chosen from any naturally occurring amino acid; (k) n=3and at least one X=Gly wherein the remaining amino acid residue(s) X areindependently chosen from any naturally occurring amino acid; (1) n=3and at least two X=Gly wherein the remaining amino acid residue(s) X areindependently chosen from any naturally occurring amino acid; (m) n=2and each X=Ala or Gly; (n) n=3 and each X=Ala or Gly; (o) n=3 and atleast one X=Ala or Gly wherein remaining amino acid residue(s) X areindependently chosen from any naturally occurring amino acid; or (p) n=3and at least two X=Ala or Gly wherein the remaining amino acidresidue(s) X are independently chosen from any naturally occurring aminoacid, e.g., A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA orGAG. The present invention also provides a PD1 binder (e.g., an ISVDsuch as a Nanobody) comprising one or mutations at position 11, 89, 110and 112 and an amino acid sequence having at least 85% (e.g., 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9 or 100%)sequence identity with the amino acid sequence set forth in a memberselected from the group consisting of SEQ ID NOs: 9-40 (in which anyC-terminal extension that may be present as well as any CDRs are nottaken into account for determining the degree of sequence identity). Thepresent invention also provides a multispecific immunoglobulincomprising a PD1 binder (e.g., an ISVD such as a Nanobody) that binds toPD1 linked to one or more molecules that bind to an epitope that is notthe epitope to which the PD1 binder binds (e.g., PD1, CTLA4, LAG3, BTLAand/or CD27), e.g., comprising a PD1 binding moiety and a CTLA4 bindingmoiety; a PD1 binding moiety and a BTLA binding moiety; a PD1 bindingmoiety and a LAG3 binding moiety; or a PD1 binding moiety, a LAG3binding moiety and a BTLA binding moiety, optionally linked via one ormore linkers, e.g., peptide linkers. In one embodiment of the invention,the multispecific immunoglobulin comprises a first PD1 Nanobody linkedto one or more molecules selected from the group consisting of an CTLA4Nanobody, an LAG3 Nanobody, an BTLA Nanobody, an CD27 Nanobody and anPD1 Nanobody that binds to the same or a different epitope as that ofthe first PD1 Nanobody; e.g., comprising a PD1 binding moiety and aCTLA4 binding moiety; a PD1 binding moiety and a BTLA binding moiety; aPD1 binding moiety and a LAG3 binding moiety; or a PD1 binding moiety, aLAG3 binding moiety and a BTLA binding moiety.

The present invention includes a polypeptide, ISVD or Nanobodycomprising an amino acid sequence that is described herein, e.g., whichis selected from SEQ ID NOs: 9-40.

The present invention also provides a PD1 binder of the presentinvention (e.g., an immunoglobulin single variable domain (ISVD) ormultispecific ISVD such as a Nanobody) in association with a furthertherapeutic agent.

Injection devices and vessels comprising PD1 binder (e.g.,immunoglobulin single variable domain (ISVD) or multispecific ISVD suchas a Nanobody) optionally in association with a further therapeuticagent are provided by the present invention.

The present invention provides a polynucleotide encoding a PD1 binder(e.g., immunoglobulin single variable domain (ISVD) or multispecificISVD such as a Nanobody); or a vector comprising the polynucleotide or ahost cell comprising the polynucleotide or vector.

The present invention provides a method for making an PD1 binder (e.g.,immunoglobulin single variable domain (ISVD) or multispecific ISVD suchas a Nanobody) comprising introducing a polynucleotide encoding theimmunoglobulins into a host cell and culturing the host cell in a mediumunder conditions favorable to expression of said immunoglobulin fromsaid polynucleotide and, optionally, purifying the immunoglobulin fromsaid host cell and/or said medium. PD1 binders (e.g., an ISVDs such as aNanobodies) produced by such methods are part of the present invention.

The present invention also provides a method for preventing PD1 frombinding to PD-L1 and/or PD-L2 comprising contacting said PD1 with a PD1binder (e.g., immunoglobulin single variable domain (ISVD) ormultispecific ISVD such as a Nanobody) optionally in association with afurther therapeutic agent.

The present invention also provides a method for enhancing an immuneresponse in the body of a subject comprising administering an effectiveamount of a PD1 binder of the present invention (e.g., immunoglobulinsingle variable domain (ISVD) or multispecific ISVD such as a Nanobody)to the subject optionally in association with a further therapeuticagent. In addition, the present invention also provides a method fortreating or preventing cancer (e.g., metastatic cancer, a solid tumor, ahematologic cancer, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma,neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer,bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breastcancer, prostate cancer, bone cancer, lung cancer, non-small cell lungcancer, gastric cancer, colorectal cancer, cervical cancer, synovialsarcoma, head and neck cancer, squamous cell carcinoma, multiplemyeloma, renal cell cancer, retinoblastoma, hepatoblastoma,hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney,Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma,pituitary adenoma, vestibular schwannoma, a primitive neuroectodermaltumor, medulloblastoma, astrocytoma, anaplastic astrocytoma,oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemiavera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma,thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer,breast cancer or gastric cancer) or an infectious disease (bacterialinfection, a viral infection or a fungal infection) in the body of asubject comprising administering an effective amount of PD1 binder(e.g., immunoglobulin single variable domain (ISVD) or multispecificISVD such as a Nanobody) optionally in association with a furthertherapeutic agent to the subject. In an embodiment of the invention, thesubject is also administered a further therapeutic agent or atherapeutic procedure in association with the PD1 binder.

The present invent invention provides PD1 binders (e.g., ISVDs such asNanobodies) that bind to PD1 comprising: CDR1 comprising the amino acidsequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); CDR2comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4;wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V);and CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5,wherein X is W or F), which comprise one or more mutations at position11 (e.g., L11V) and 89 (e.g., I89L) or one or more mutations selectedfrom E1D, L11V, A14P, W52aV, N73(Q, P or S), A74S, K83R, I89L, W100aFwith respect to SEQ ID NO: 1 or 2. In an embodiment of the invention,the mutations are E1D, L11V, A14P, K83R and I89L; or L11V, A14P, K83Rand I89L. In an embodiment of the invention, the mutations are E1D,L11V, A14P, W52aV, N73(Q, P or S), A74S, K83R, I89L, W100aF orL11V,A14P,W52aV, N73(Q, P or S), A74S, K83R, I89L, W100aF. In anembodiment of the invention, the PD1 binder comprises the amino acidsequence: DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAVITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAGDK HQSSWYDYWGQGTLVTVSS (SEQ ID NO: 57).

The present invention also provides LAG3 binders that bind to LAG3comprising CDR1 comprising the amino acid sequence GRTFSDYVMG (SEQ IDNO: 65); CDR2 comprising the amino acid sequence AISESGGRTHYADXKG (SEQID NO:66; wherein X is A or S or AISESGGRTH (SEQ ID NO: 139, amino acids1-10 of SEQ ID NO: 66); and CDR3 comprising the amino acid sequenceTLLWWTSEYAPIKANDYDY (SEQ ID NO: 67), e.g., comprising the amino acidsequence: EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKERE FVAAISESGGRTHYADSVKG RFTISRDNSK NTLYLQMNSL RPEDTALYYC ATTLLWWTSE YAPIKANDYDYWGQGTLVTV SS (SEQ ID NO: 64).

The PD1 and LAG3 binders may be in a single molecule such as a PD1/LAG3binder, which is part of the present invention, that binds to PD1 andLAG3 that comprises: a PD1 binder comprising: CDR1 comprising the aminoacid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); CDR2comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4;wherein X is W or V) or VITXSGGITY (SEQ ID NO: 7; wherein X is W or V);and CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5,wherein X is W or F); and a LAG3 binder comprising: CDR1 comprising theamino acid sequence GRTFSDYVMG (SEQ ID NO: 65); CDR2 comprising theamino acid sequence AISESGGRTHYADXKG (SEQ ID NO: 66; wherein X is A orS) or AISESGGRTH (SEQ IDNO: 139, amino acids 1-10 of SEQ ID NO: 66); andCDR3 comprising the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ ID NO:67); and, optionally, a half-life extender, e.g., wherein the PD1 bindercomprises the amino acid sequence: DVQLVESGGG VVQPGGSLRL SCAASGSIASIHAMGWFRQA PGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPEDTALYYCAGDK HQSSWYDYWG QGTINTVSS; and (SEQ ID NO: 57); and the LAG3binder comprises the amino acid sequence: EVQLVE SGGGVVQPGG SLRLSCAASGRTFSDYVMGW FRQAPGKERE FVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSLRPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS (SEQ ID NO: 64); and,optionally, a half-life extender. For example, in an embodiment of theinvention, the PD1/LAG3 binder comprises the moieties, e.g., in theorder shown:

-   -   the PD1 binder 102C12 (E1D,L11V,A14P,A74S,K83R,I89L) or        1PD102C12 (E1D, L11V, A14P, W52aV, N73X (e.g., N73P or N73Q or        N73S), A74S, K83R, I89L, W100aF);    -   a peptide linker such as 9GS, 20GS or 35 GS (e.g.,        GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));    -   the LAG3 binder 11B09 or 11B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L);    -   a peptide linker such as 9GS, 20GS or 35 GS (e.g.,        GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));    -   a half-life extender such as ALB11002; and, optionally,    -   a C-terminal extension such as an alanine;    -   or    -   the PD1 binder 102C12 (E1D,L11V,A14P,A74S,K83R,I89L) or        1PD102C12 (E1D, L11V, A14P, W52aV, N73X (e.g., N73P or N73Q or        N73S), A74S, K83R, I89L, W100aF);    -   a peptide linker such as 9GS, 20GS or 35 GS (e.g.,        GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));    -   the PD1 binder 102C12 (L11V,A14P,A74S,K83R,I89L) or 1PD102C12        (L11V, A14P, W52aV, N73X (e.g., N73P or N73Q or N73S), A74S,        K83R, I89L, W100aF);    -   a peptide linker such as 9GS, 20GS or 35 GS (e.g.,        GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));    -   the LAG3 binder 11B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L);    -   a peptide linker such as 9GS, 20GS or 35 GS (e.g.,        GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));    -   the LAG3 binder 11B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L);    -   a peptide linker such as 9GS, 20GS or 35 GS (e.g.,        GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70));    -   a half-life extender such as ALB11002; and, optionally,    -   a C-terminal extension such as an alanine.

The half -life extender is, in an embodiment of the invention, a humanserum albumin (HSA) binder such as ALB11002. In an embodiment of theinvention, the HSA binder comprises CDR1 comprising the amino acidsequence GFTFSSFGMS (SEQ ID NO: 60); CDR2 comprising the amino acidsequence SISGSGSDTL (SEQ ID NO: 152; amino acids 1-10 of SEQ ID NO: 61);and CDR3 comprising the amino acid sequence GGSLSR (SEQ ID NO: 62),e.g., comprising the amino acid sequence EVQLVESGGG VVQPGNSLRLSCAASGFTFSS FGMSWVRQA PGKGLEWVSS ISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPED TALYYCTIGG SLSRSSQGTL VTVSSA (SEQ ID NO: 142). Such half-lifeextenders themselves are part of the present invention.

In an embodiment of the invention, the PD1 binder, LAG3 binder, HSAbinder and/or PD1/LAG3 binder is in an injection device or vesseloptionally in association with a further therapeutic agent. Such adevice or vessel is part of the present invention.

The present invention also provides a polynucleotide encoding any of thebinders set forth herein as well as any vector (e.g., plasmid)comprising the polynucleotide as well as any host cell (e.g., CHO orfungal cell such as Pichia, e.g., P. pastoris) comprising thepolynucleotide or vector, e.g., ectopic or integrated into one or morehost cell chromosomes.

The present invention also provides a method for making any of thebinders set forth herein comprising introducing a polynucleotideencoding the binder into a host cell (e.g., CHO or fungal cell such asPichia, e.g., P. pastoris) and culturing the host cell in a medium underconditions favorable to expression of said binder from saidpolynucleotide and, optionally, purifying the binder from said host celland/or said medium. Optionally, the vector or polynucleotide isintegrated into one or more host cell chromosomes. Any binder producedby such a method is also part of the present invention.

The present invention also provides a method for preventing PD1 frombinding to PD-L1 and/or PD-L2 or for inhibiting any PD1 activity, e.g.,as discussed herein, comprising contacting said PD1 with a PD1 binder ofthe present invention (e.g., a PD1/LAG3 binder) optionally inassociation with a further therapeutic agent. The present invention alsoprovides a method for preventing LAG3 from binding to MHC class II orfor inhibiting any PD1 activity, e.g., as discussed herein comprisingcontacting said LAG3 with a LAG3 binder of the present invention (e.g.,a PD1/LAG3 binder) optionally in association with a further therapeuticagent. The present invention also provides a method for enhancing animmune response in the body of a subject (e.g., a human) comprisingadministering an effective amount of binder of the present invention(e.g., PD1/LAG3 binder) to the subject optionally in association with afurther therapeutic agent. The present invention also provides a methodfor treating or preventing cancer (e.g., metastatic cancer, a solidtumor or a hematologic cancer) or an infectious disease (e.g., bacterialinfection, a viral infection or a fungal infection) in the body of asubject (e.g., a human) comprising administering an effective amount ofbinder of the present invention (e.g., a PD1/LAG3 binder) optionally inassociation with a further therapeutic agent to the subject. Whenadministered to a subject, a PD1 and/or LAG3 binder of the presentinvention (e.g., a PD1/LAG3 binder) is optionally administered inassociation with a further therapeutic agent or a therapeutic proceduree.g., surgical tumorectomy.

DESCRIPTION OF THE FIGURES

FIG. 1. A table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT)

FIG. 2(1-5). PD1 binder sequences.

FIG. 3(A-B). Alignment of 102C12 sequence with that of the SEQ ID NOs:9-40 (see WO 2008/071447, SEQ ID NO: 348).

FIG. 4. Predominant N-linked glycans for monoclonal antibodies producedin Chinese hamster ovary cells (CHO N-linked glycans) and in engineeredyeast cells (engineered yeast N-linked glycans): squares:N-acetylglucosamine (GlcNac); circles: mannose (Man); diamonds:galactose (Gal); triangles: fucose (Fuc).

FIG. 5. Monovalent binding of CHO-K1 expressing human PD1 by F023700275(1PD102C12(A14P,A74S,K83R)-FLAG3-HIS6) or F023700706 (1PD102C12(L11V,A14P,A74S,K83R,I89L)-FLAG3-HIS6). GAM=goat anti-mouse secondaryantibody use to detect mouse antibody that binds to FLAG epitope;α-FLAG+GAM=goat anti-mouse secondary antibody used plus mouse antibodythat binds to FLAG epitope that is at C-terminus of the construct.

FIG. 6. Monovalent binding of 3A9 cells expressing human LAG3 byF0237611B09-FLAG-His6 or F023700842(F0237611B09(L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-FLAG3-HIS6.

FIG. 7(A-B). Binding of (A) CHO expressing human PD-1 by F023700931,F023700924, F023700933 or F023700962 or (B) 3A9 expressing rhesus PD-1by F023700931 (Pichia or CHO expressed), F023700924 (Pichia or CHOexpressed), F023700933, F023700962, F023700678 (1PD102C12(A14P,A745,K83R)-35G5-ALB11002) or F023701127. ABH0074+GAM=goatanti-mouse secondary antibody used plus mouse antibody ABH0074 thatbinds to nanobody frameworks.

FIG. 8(A-B). Binding of (A) CHO expressing human LAG3 by F023700931,F023700924 or F023700962 or (B) 3A9 expressing rhesus LAG3 by F023700931(Pichia or CHO expressed), F023700924 (Pichia or CHO expressed),F023700933 or F023700962.

FIG. 9(A-H). Blockage of binding between (A) human PD-L1-Fe and CHO-K1expressing human PD1 by F023700275 or F023700706; (B) human PD-L2-Fc andCHO-K1 expressing human PD1 by F023700275 or F023700706; (C) humanPD-L1-Fc and CHO-K1 expressing human PD1 by F023700931 (Pichia or CHOexpressed), F023700924 (Pichia or CHO expressed), F023700933 orF023700962; (D) human PD-L2-Fc and CHO-K1 expressing human PD1 byF023700931 (Pichia or CHO expressed), F023700924 (Pichia or CHOexpressed), F023700933 or F023700962; (E) human PD-L1-Fc and CHO-K1expressing human PD1 by F023700931, F023700924, F023700933 orF023700962; (F) human PD-L2-Fc and CHO-K1 expressing human PD1 byF023700931, F023700924, F023700933 or F023700962; (G) human PD-L1-Fe andCHO-K1 expressing human PD1 by F023700929 (1PD102C12(L11V,A14P,A74S,K83R,I89L)-HIS6), F023701190 (1PD102C12(E1D,L11V,A14P,W52aV,A74S,K83R,I89L,W100aF)-HIS6), F023701192 (1PD102C12(E1D,L11V,A14P,W52aV,N73Q,A74S,K83R,I89L,W100aF)-HIS6, or F023701193(1PD102C12(E1D,L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100aF)-HIS6); (H)human PD-L2-Fc and CHO-K1 expressing human PD1 by F023700929 (1PD102C12(L11V,A14P,A74S,K83R,I89L)-HIS6), F023701190 (1PD102C12(E1D,L11V,A14P,W52aV,A74S,K83R,I89L,W100aF)-HIS6), F023701192 (1PD102C12(E1D,L11V,A14P,W52aV,N73Q,A74S,K83R,I89L,W100aF)-HIS6, or F023701193(1PD102C12 (E1D,L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100aF)-HIS6).US=Stained; hPD-L1 EC30=the staining intensity of hPD-L1-Fe that hasbeen titrated to give 30% of the maximal staining intensity that couldbe obtained; GAH=goat anti-human secondary antibody used to detect thehuman Fc portion of hPD-L1-Fe.

FIG. 10(A-C). Blockade of human LAG3-Fc binding to Daudi cells by (A)F0237611B09-FLAG3-H156, F023700842-FLAG3-HIS6 or human LAG3-Fc; (B)F023700931 (Pichia or CHO expressed), F023700924 (Pichia or CHOexpressed), F023700933 or F023700962, human LAG3-Fc; or (C) F023700924,F023700931 or F023700962 or human LAG3-Fc. Sec only=secondary antibodyonly (GAH/GAM).

FIG. 11(A-B). Proximity Assay (beta-galactosidase enzyme fragmentcomplementation assay system) with (A) F023700931; F023701016,F023701017, control Nanobody (IRR00085; respiratory syncitia virus (RSV)binder), F023700933 or F023700962; or (B) F023700924; F023700969,F023700970, control Nanobody (IRR00085), F023700933 or F023700962.

FIG. 12(A-E). Activation of Jurkat T-cells (expression of luciferaseoperably linked to IL2 promoter) in presence of HSA by (A) F023700706 orcontrol Nanobody (IRR00043; two anti-lysozyme nanobodies linked with a35GS linker that has a C-terminal FLAG3-His6), (B) F023700931 (Pichia orCHO expressed), F023700924 (Pichia or CHO expressed), F023700933,F023700962 or control Nanobody (IRR00085 or IRR00087; RSV binder), (C)F023700924 F023700969, F023700970 or control Nanobody (IRR00043), (D)F023700931, F023701016, F023701017 or control Nanobody (IRR00043), or(E) F023700706, F023701192 (1PD102C12(E1D,L11V,A14P,W52aV,N73Q,A74S,K83R,I89L,W100aF)-HIS6, F023701193 (1PD102C12 (E1D,L11V,A14P,W52aV,N73P,A74S,K83R,I89L,W100aF)-HIS6 or control nanobody (IRR00088; RSVbinder).

FIG. 13(A-R). Activation of human peripheral blood monocytes (IL2production) from donors (A) 91,(B) 985, (C) 907, (D) 91, (E) 985, (F)907, (G) 91 (with 10 nM SEB), (H) 985 (with 10 nM SEB), (I) 907 (with 10nM SEB), (J) 91(with 25 nM SEB), (K) 985 (with 25 nM SEB), (L) 907 (with25 nM SEB) with F023700931 (Pichia or CHO expressed), F023700924 (Pichiaor CHO expressed), F023700933, F023700962 or control Nanobody; or (M) 91(N) 985 (O) 907 with F023700931, F023700924 or control Nanobody; or (P)91, (Q) 985 or (R) 907 with F023700924, F023700969, F023700970.

FIG. 14(A-F). Mixed lymphocyte assay of CD4 T-cells and dendritic cellsfrom different donors determining (A-C) interferon-gamma production atvarying concentrations of Nanobody F023700931 (Pichia or CHO expressed),F023700924 (Pichia or CHO expressed) or F023700933 or a controlantibody; (D-F) interferon-gamma production at varying concentrations ofNanobody F023700924, F023700969, F023700970, F023700931 F023701016 orF023701017 or control Nanobody.

FIG. 15(A-D). Activation assay of 3A9 T-cells expressing human LAG3 inthe presence of HSA and in the presence of (A) F023700656 (11B09 (E1D)),F023700842 or control IgG4; (B) F023700931 (Pichia or CHO expressed),F023700924 (Pichia or CHO expressed), F023700933 or F023700962 orcontrol Nanobody (IRR00085 or IRR00087; RSV binders); (C) F023700924,F023700969, F023700970 or control Nanobody; (D) F023700931, F023701016,or F023701017 or control Nanobody.

FIG. 16(A-B). Activation of Jurkat T-cells expressing human LAG3 andhuman PD1 and Raji antigen-presenting cells in the presence of (A)F023700931 (Pichia or CHO expressed), F023700924 (Pichia or CHOexpressed), F023700933 or F023700862 [should this be F023700892] orcontrol Nanobody or (B) F023700924, F023700969, F023700970, F023700931,F023701016, F023701017, F023700933 or F023700962 or control Nanobody.

FIG. 17(A-C). Human T-cell clone activation (interferon-gammaproduction) in the presence of JY cells expressing human PDL1 in thepresence of varying concentrations of (A) F023700931 (Pichia or CHOexpressed), F023700924 (Pichia or CHO expressed), F023700933 orF023700962 or control Nanobody; (B) F023700924, F023700969, F023700970,F023700933 or F023700962 or control Nanobody; or (C) F023700931,F023701016, F023701017, F023700933 or F023700962 or control Nanobody.

FIG. 18(A-P). Sequences of the present invention.

FIG. 19(A-I). Serum preAb reactivity to F023700924 and F023700931 and atrivalent control Nanobody T013700112 (lacking mutations to reducepre-existing antibody binding) by healthy human subject sera at (A) 125seconds and (B) 360 seconds; by cancer patient sera at (C) 125 secondsor (D) 360 seconds; or by sera of patients suffering from (E) melanoma,(F) non-small cell lung cancer (NSCLC), (G) head & neck cancer, (H)gastric cancer or (I) colorectal cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides ISVDs that comprise mutations which blockreactivity of pre-existing antibodies (pre-antibodies) to neo-epitopeswithin the ISVDs. Neoepitopes are epitopes within a protein which arerevealed when the protein is mutated (e.g., truncated) or its folding isaltered. Pre-existing antibodies are antibodies existing in the body ofa patient prior to receipt of an ISVD. The ISVDs of the presentinvention are based, in part, in llama antibodies whose C-terminalconstant domains have been removed; thus, exposing the neo-epitopes inthe C-terminus of the resulting VHH to pre-antibody binding. It has beendiscovered that the combination of mutations of residues 11 and 89(e.g., L11V and I89L or V89L) led to a surprising lack of pre-antibodybinding. Mutations in residue 112 have also been shown to remarkablyreduce pre-antibody binding. Buyse & Boutton (WO2015/173325) includeddata showing that the combination of an L 11V and V89L mutation provideda remarkable improvement in reducing pre-antibody binding compared to anL11V mutation alone or a V89L mutation alone. For example, Table H ofBuyse & Boutton on page 97 showed comparative data for an ISVD with aV89L mutation alone (with or without C-terminal extension) and the sameISVD with a V89L mutation in combination with an L11V mutation (again,with or without a C-terminal extension). Also, although generated in twoseparate experiments, the data shown in Table H for the L11V/V89Lcombination as compared to the data given in Table B for an L11Vmutation alone (in the same ISVD) showed that the pre-antibody bindingreduction that is obtained by the L11V/V89L combination was greater thanthat for the L11V mutation alone. Since the llama antibody scaffoldstructure is known to be very highly conserved, the effect of themutations at positions 11 and 89 is very likely to exist for any ISVD.Indeed, the effect was demonstrated, in FIG. 19, with the instantbinders, F023700924 and F023700931, which were shown to exhibit very lowlevels of pre-antibody binding in healthy subjects and subjectssuffering from cancer.

In the present application, the amino acid residues/positions in animmunoglobulin heavy-chain variable domain will be indicated with thenumbering according to Kabat. For the sake of convenience, FIG. 1 givesa table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT). This point is alsofurther discussed herein.

With regards to the CDRs, as is well-known in the art, there aremultiple conventions to define and describe the CDRs of a VH or VHHfragment, such as the Kabat definition (which is based on sequencevariability and is the most commonly used) and the Chothia definition(which is based on the location of the structural loop regions).Reference is for example made to the website www.bioinf.org.uk/abs/. Forthe purposes of the present specification and claims, even though theCDRs according to Kabat may also be mentioned, the CDRs are mostpreferably defined on the basis of the Abm definition (which is based onOxford Molecular's AbM antibody modelling software), as this isconsidered to be an optimal compromise between the Kabat and Chotiadefinitions. Reference is again made to the websitewww.bioinf.org.uk/abs/). See Sequences of Proteins of ImmunologicalInterest, Kabat, et al.; National Institutes of Health, Bethesda, Md.;5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem.32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, etal., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature342:878-883; Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Elvin A.Kabat, Tai Te Wu, Carl Foeller, Harold M. Perry, Kay S. Gottesman (1991)Sequences of Proteins of Immunological Interest; Protein Sequence andStructure Analysis of Antibody Variable Domains. In: AntibodyEngineering Lab Manual (Ed.: Duebel, S. and Kontermann, R.,Springer-Verlag, Heidelberg). In an embodiment of the invention, CDRdetermination is according to Kabat, e.g., wherein FR1 of a VHHcomprises the amino acid residues at positions 1-30, CDR1 of a VHHcomprises the amino acid residues at positions 31-35, FR2 of a VHHcomprises the amino acids at positions 36-49, CDR2 of a VHH comprisesthe amino acid residues at positions 50-65, FR3 of a VHH comprises theamino acid residues at positions 66-94, CDR3 of a VHH comprises theamino acid residues at positions 95-102, and FR4 of a VHH comprises theamino acid residues at positions 103-113.

In an embodiment of the invention, CDRs are determined according toKontermann and Dübel (Eds., Antibody Engineering, vol 2, Springer VerlagHeidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010).

The term “immunoglobulin single variable domain” (also referred to as“ISV” or ISVD”) is generally used to refer to immunoglobulin variabledomains (which may be heavy chain or light chain domains, including VH,VHH or VL domains) that can form a functional antigen binding sitewithout interaction with another variable domain (e.g. without a VH/VLinteraction as is required between the VH and VL domains of conventional4-chain monoclonal antibody). Examples of ISVDs will be clear to theskilled person and for example include Nanobodies (including a VHH, ahumanized VHH and/or a camelized VHs such as camelized human VHs),IgNAR, domains, (single domain) antibodies (such as dAbs™) that are VHdomains or that are derived from a VH domain and (single domain)antibodies (such as dAbs™) that are VL domains or that are derived froma VL domain. ISVDs that are based on and/or derived from heavy chainvariable domains (such as VH or VHH domains) are generally preferred.Most preferably, an ISVD will be a Nanobody.

The term “Nanobody” is generally as defined in WO 08/020079 or WO09/138519, and thus in a specific aspect generally denotes a VHH, ahumanized VHH or a camelized VH (such as a camelized human VH) orgenerally a sequence optimized VHH (such as e.g. optimized for chemicalstability and/or solubility, maximum overlap with known human frameworkregions and maximum expression). It is noted that the terms Nanobody orNanobodies are registered trademarks of Ablynx N.V. and thus may also bereferred to as Nanobody® and/or Nanobodies®). An example of an ISVD is102C12 (E1D, L11V, A14P, A74S, K83R, I89L). Other ISVDs also appear inTables A and C herein.

A multispecific binder (e.g., multispecific ISVD) is a molecule thatcomprises, for example, a first PD1 or LAG3 binding moiety (e.g., anISVD or a Nanobody) and one or more (e.g., 1, 2, 3, 4, 5) additionalbinding moieties (e.g., an ISVD or a Nanobody) that bind to an epitopeother than that of the first PD1 or LAG3 binding moiety (e.g., to CTLA4,CD27 and/or BTLA); e.g., comprising a PD1 or LAG3 binding moiety and aCTLA4 binding moiety; a PD1 or LAG3 binding moiety and a BTLA bindingmoiety; one or two PD1 binding moieties and one or two LAG3 bindingmoieties and a human serum albumin binding moiety; or a PD1 bindingmoiety, a LAG3 binding moiety and a BTLA binding moiety. A multispecificbinder is, for example, F023700931 or F023700899.

A binding moiety or binding domain or binding unit is a molecule such asan ISVD or Nanobody that binds to an antigen. A binding moiety orbinding domain or binding unit may be part of a larger molecule such asa multivalent or multispecific immunoglobulin that includes more thanone moiety, domain or unit and/or that comprises another functionalelement, such as, for example, a half-life extender (HLE), targetingunit and/or a small molecule such a polyethyleneglycol (PEG).

A monovalent PD1 or LAG3 binder (e.g., ISVD such as a Nanobody) is amolecule that comprises a single antigen binding domain. A bivalent PD1or LAG3 binder comprises two antigen binding domains (e.g., conventionalantibodies including bispecific antibodies). A multivalent PD1 or LAG3binder comprises more than one antigen-binding domain.

A monospecific PD1 or LAG3 binder binds a single antigen; a bispecificPD1 or LAG3 binder binds to two different antigens and a multispecificPD1 or LAG3 binder binds to more than one antigen.

A biparatopic PD1 or LAG3 binder is monospecific but binds to twodifferent epitopes of the same antigen. A multiparatopic PD1 or LAG3binder binds the same antigen but to more than one epitope in theantigen.

The term “half-life” as used herein relation to a PD1 and/or LAG3 binderor ISVD, Nanobody, ISVD-based biological, Nanobody-based biological orany other polypeptide referred to herein can generally be defined asdescribed in paragraph o) on page 57 of WO 08/020079 and as mentionedtherein refers to the time taken for the serum concentration of thepolypeptide to be reduced by 50%, in vivo, for example due todegradation of the polypeptide and/or clearance or sequestration of thepolypeptide by natural mechanisms. The in vivo half-life of apolypeptide of the invention can be determined in any manner known perse, such as by pharmacokinetic analysis. Suitable techniques will beclear to the person skilled in the art, and may for example generally beas described in paragraph o) on page 57 of WO 08/020079. As alsomentioned in paragraph o) on page 57 of WO 08/020079, the half-life canbe expressed using parameters such as the t½-alpha, t½-beta and the areaunder the curve (AUC). In this respect it should be noted that the term“half-life” as used herein in particular refers to the t½-beta orterminal half-life (in which the t½-alpha and/or the AUC or both may bekept out of considerations). Reference is for example made to theExperimental Part below, as well as to the standard handbooks, such asKenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook forPharmacists and Peters et al, Pharmacokinete analysis: A PracticalApproach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi& D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).Similarly, the terms “increase in half-life” or “increased half-life” asalso as defined in paragraph o) on page 57 of WO 08/020079 and inparticular refer to an increase in the t½-beta, either with or withoutan increase in the t½-alpha and/or the AUC or both.

When a term is not specifically defined herein, it has its usual meaningin the art, which will be clear to the skilled person. Reference is forexample made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd.Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10th Ed. Blackwell Publishing, UK (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein.

For a general description of multivalent and multispecific polypeptidescontaining one or more Nanobodies and their preparation, reference isalso made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350,2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001),277-302; as well as to for example WO 96/34103, WO 99/23221, WO04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO2009/068627.

“Isolated” PD1 and/or LAG3 binders (e.g., an ISVD such as a Nanobody),polypeptides, polynucleotides and vectors, are at least partially freeof other biological molecules from the cells or cell culture from whichthey are produced. Such biological molecules include nucleic acids,proteins, lipids, carbohydrates, or other material such as cellulardebris and growth medium. An “isolated” PD1 and/or LAG3 binder mayfurther be at least partially free of expression system components suchas biological molecules from a host cell or of the growth mediumthereof. Generally, the term “isolated” is not intended to refer to acomplete absence of such biological molecules or to an absence of water,buffers, or salts or to components of a pharmaceutical formulation thatincludes the antibodies or fragments.

The phrase “control sequences” refers to polynucleotides necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid or polynucleotide is “operably linked” when it is placedinto a functional relationship with another polynucleotide. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,but not always, “operably linked” means that the DNA sequences beinglinked are contiguous, and, in the case of a secretory leader,contiguous and in reading phase. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice. The present invention includes polynucleotidesencoding the PD1 and/or LAG3 binders which, optionally, are operablylinked to one or more control sequences such as a promoter.

A “PD1 and/or LAG3” binder refers to a binder that includes

-   -   a PD1 binder; or    -   a LAG3 binder; or    -   both a PD1 binder and a LAG3 binder (i.e., a “PD1/LAG3 binder”)        and, optionally, to another binder that binds, for example,        human serum albumin. In an embodiment of the invention, a        PD1/LAG binder is    -   102C12 (E1D,L11V,A14P,A74S,K83R,I89L)-35GS-11B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-ALB11002-A; or    -   102C12 (E1D,L11V,A14P,A74S,K83R,I89L)-35GS-102C12        (L11V,A14P,A74S,K83R,I89L)-35GS-11B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-11B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-ALB11002-A;        as set forth herein.    -   See F023700924 or F023700931 herein. A PD1/LAG3 binder includes        a PD1 binder and a LAG3 binder.

The scope of the present invention includes any PD1 binder set forth inFIG. 18(A-P) (or any PD1 binder comprising CDR1, CDR2 and CDR3 of such aPD1 binder), any LAG3 binder set forth in FIG. 18(A-P) (or any LAG3binder comprising CDR1, CDR2 and CDR3 of such a LAG3 binder) or anyPD1/LAG3 binder set forth in FIG. 18(A-P) or any PD1/LAG3 bindercomprising CDR1, CDR2 and CDR3 of the PD1 binding moiety and/or LAG3binding moiety thereof. The binders set forth in FIG. 18(A-P), in anembodiment of the invention, do not include the C-terminal extender(e.g., A), FLAG and/or HIS tags therein (e.g., HHHHHH (amino acids 29-34or SEQ ID NO: 68); AAAHHHHHH (SEQ ID NO: 69); orAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH (SEQ ID NO: 68)). Any such binder orCDR may, in an embodiment may be a variant of what is set forth in FIG.18(A-P), e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 pointmutations (e.g., conservative substitutions or deletions).

In general, the basic antibody structural unit comprises a tetramer.Each tetramer includes two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of the heavy chain maydefine a constant region primarily responsible for effector function.Typically, human light chains are classified as kappa and lambda lightchains. Furthermore, human heavy chains are typically classified as mu,delta, gamma, alpha, or epsilon, and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavychains, the variable and constant regions are joined by a “J” region ofabout 12 or more amino acids, with the heavy chain also including a “D”region of about 10 more amino acids. See generally, FundamentalImmunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

Examples of antigen-binding fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, and Fv fragments, and single-chain Fv molecules.

A “PD1 binder” or “PD1 ISVD” or “PD1 nanobody” refers to a binder orISVD or Nanobody, respectively, that binds to PD1. A similar conventionmay be applied with respect to molecules that bind to LAG3 or CTLA4 oranother antigen.

The following properties are associated with the indicated mutations inthe PD1 binder 102C12:

E1D: Prevent pyroglutamic acid formation in the first amino acid of theconstruct E1

L11V: Decrease pre-antibody binding

A14P: Humanization

W52aV: Prevent oxidation of W52a

N73P: Prevent N73 deamidation

N73Q: Prevent N73 deamidation

N73 S: Prevent N73 deamidation

A74S: Humanization

K83R: Humanization

I89L: Decrease pre-antibody binding

W100aF: Prevent oxidation of W100a or the LAG3 binder 11B09:

L11V: Decrease pre-antibody binding

A14P: Humanization

R41P : Humanization

N43K: Humanization

A62S: Humanization

A74S: Humanization

K83R: Humanization

V89L: Decrease pre-antibody binding

In an embodiment of the invention, PD1 is human PD1. In an embodiment ofthe invention, human PD1 comprises the amino acid sequence:

(SEQ ID NO: 137) MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPALLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLAAFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGTYLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSPRPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTIGARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVPCVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL

In an embodiment of the invention, LAG3 is human LAG3. In an embodimentof the invention, human LAG3 comprises the amino acid sequence:

(SEQ ID NO: 138) MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPAQLPCSPTIPL QDLSLLRRAG VTWQHQPDSG PPAAAPGHPLAPGPHPAAPS SWGPRPRRYT VLSVGPGGLR SGRLPLQPRVQLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRALSCRLRLRLGQAS MTASPPGSLR ASDWVILNCS FSRPDRPASVHWFRNRGQGR VPVRESPHHH LAESFLFLPQ VSPMDSGPWGCILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSRVGLPCRLPAGVGT RSFLTAKWTP PGGGPDLLVT GDNGDFTLRLEDVSQAQAGT YTCHIHLQEQ QLNATVTLAI ITVTPKSFGSPGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEAQEAQLLSQPW QCQLYQGERL LGAAVYFTEL SSPGAQRSGRAPGALPAGHL LLFLILGVLS LLLLVTGAFG FHLWRRQWRPRRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL

PD1 Binders

The present invention provides improved PD1 binders, for example,improved PD1 ISVDs and more in particular improved PD1 Nanobodies. Theimproved PD1 binders provided by the invention are also referred toherein as the “PD1 binders of the invention” or “PD1 binders”.

When discussed herein, monovalent PD1 binders of the present inventionwill include the amino acid sequence of SEQ ID NO: 1 or 2, but includingone or more mutations at position 1, 11, 14, 52a, 73, 74, 83, 89, 100a,110 or 112 (or at any of the mutational positions set forth herein withregard to PD1 binders of the invention).

When discussed herein, multispecific PD1 binders, for example, thatinclude a LAG3, will include PD1 binding moieties including CDR1, CDR2and CDR3 that are in the PD1 binders set forth below in Table A-1 andA-2 (e.g., in 102C12 or reference A). Optionally, the PD1 binding moietyof the multispecific binder comprises the amino acid of SEQ ID NO: 1 or2 but including one or more mutations at position 1, 11, 14, 52a, 73,74, 83, 89, 100a, 110 or 112.

The present invention provides improved PD1 binders, for example,improved PD1 ISVDs and more in particular improved PD1 Nanobodies. PD1binders of the present invention include polypeptides which are variantsof polypeptides comprising the amino acid sequence of SEQ ID NO: 1 or 2which is mutated at position 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110and/or 112. The improved PD1 binders provided by the invention are alsoreferred to herein as the “PD1 binders of the invention” or “PD1binders”. These terms encompass any molecule that binds to PD1 andincludes any of the molecules that bind to PD1 which are set forthherein. For example, the terms include an ISVD that comprises an aminoacid sequence set forth in a member selected from the group consistingof SEQ ID NOs: 9-40 and 57 as well as any conventional antibody orantigen-binding fragment thereof that includes an amino acid sequenceset forth in a members selected from the group consisting of SEQ ID NOs:9-40 and 57; a multispecific immunoglobulin, such as a bispecificimmunoglobulin (e.g., ISVD) that comprises an amino acid sequence setforth in a member selected from the group consisting of SEQ ID NOs: 9-40and 57, binds to PD1 and also binds to another antigen such as adifferent epitope of PD1, CD27, LAG3, CTLA4, BTLA, TIM3, ICOS, B7-H3,B7-H4, CD137, GITR, PD-L1, PD-L2, ILT1, ILT2 CEACAM1, CEACAM5, TIM3,TIGIT, VISTA, ILT3, ILT4, ILT5, ILT6, ILT7, ILT8, CD40, OX40, CD137,KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1,KIR3DL2, KIR3DL3, NKG2A, NKG2C, NKG2E, IL-10, IL-17, TSLP, e.g.,comprising a PD1 binding moiety and a CTLA4 binding moiety; a PD1binding moiety and a BTLA binding moiety; a PD1 binding moiety and aLAG3 binding moiety; or a PD1 binding moiety, a LAG3 binding moiety anda BTLA binding moiety. A multivalent PD1 binder comprises more than oneantigen-binding domain, for example more than one ISVD (one or more ofwhich bind to PD1). A monospecific PD1 binder binds a single antigen(PD1); a bispecific PD1 binder binds to two different antigens (one ofwhich is PD1) and a multispecific PD binder binds to more than oneantigen (one or more of which is PD1 and one or more of which is adifferent antigen). For example, in an embodiment of the invention, aPD1 binder comprises an amino acid sequence selected from SEQ ID NOs:106-124.

More in particular, the invention aims to provide improved PD1 bindersthat are variants of 102C12 and Reference A and that have reducedbinding by interfering factors (generally referred to as “pre-existingantibodies”) that may be present in the sera from some healthy humansubjects as well as from patients. See WO 12/175741, WO 2013/024059 andalso for example by Holland et al. (J. Clin. Immunol. 2013,33(7):1192-203) as well the PCT application PCT/EP2015/060643(WO2015/173325).

As further described herein, the PD1 binders of the invention preferablyhave the same combination of CDRs (i.e. CDR1, CDR2 and CDR3) as arepresent in 102C12 or in Reference A. WO 2008/071447 describes Nanobodiesthat can bind to PD1 and uses thereof. SEQ ID NO: 348 of WO 2008/071447disclosed a PD-1 specific Nanobody called 102C12, the sequence of whichis given herein as SEQ ID NO: 1. Also, a variant of 102C12 with ahumanizing Q108L substitution (also referred to herein as “Reference A”)is used herein as a reference compound and its sequence is given hereinas SEQ ID NO: 2. The present invention includes PD1 binders that includea mutation at position 108, e.g., Q108L.

The present invention includes PD1 binders which are variants of 102C12and PD1/LAG3 binders comprising such variants of 102C12 which are setforth below in Table A-2 below. The scope of the present inventionincludes PD1 binders that include CDR1, CDR2 and CDR3 of said varaintsset forth below in Table A-2 as well as PD1/LAG3 binders comprising aPD1 binding moiety that includes CDR1, CDR2 and CDR3 of said varaintsset forth below in Table A-2.

TABLE A-1 PD1 Binder 102C12. SEQ ID NO Description Sequence 1WO 2008/071447, EVQLVESGGGLVQAGGSLRLSCAASGSIAS IHAMG WFRQAPGKEREFVA VITSEQ ID NO: 348 WSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAG DKHQSS(102C12) (may be WYDY WGQGTQVTVSS referred to herein as ″1PD102C12″) 2reference A EVQLVESGGGLVQAGGSLRLSCAASGSIAS IHAMG WFRQAPGKEREFVA VITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAG DKHQSS WYDY WGQGTLVTVSS

Sequence Optimized Variant 102C12 PD1 Binders (optionally fused to an HSAbinder) SO 1PD102C12 monomer DVQLVESGGG VVQPGGSLRL SCAASGSIAS  IHAMGWFRQA PGKEREFVA V 102C12 (E1D, L11V,ITWSGGITYY ADSVKGRFTI SRDNSKNTVY LQMNSLRPED TALYYCAG DKA14P, A74S, K83R,  HQSSWYDY WG QGTLVTVSS 189L) Target: hPD-1SEQ ID NO: 57 SEQ ID NO: 3 PD1 binder CDR1 (Kabat) IHAMG SEQ ID NO: 4PD1 binder CDR2 (Kabat) VITXSGGITYYADSVKG wherein X is W or V(e.g., VITwSGGITYYADSVKG (SEQ ID NO: 143) or VITvSGGITYYADSVKG)(SEQ ID NO: 144)) SEQ ID NO: 5 PD1 binder CDR3  DKHQSSXYDY (Kabat/Abm) wherein X is W or F (e.g., DKHQSSwYDY (SEQ ID NO: 145) or DKHQSSfYDY)(SEQ ID NO: 146)) SEQ ID NO: 6 PD1 binder CDR1 (Abm) GSIAS IHAMGSEQ ID NO: 7 PD1 binder CDR2 (Abm) VITXSGGITY wherein X is W or V(e.g., VITwSGGITY (SEQ ID NO: 147) or VITvSGGITY) (SEQ ID NO: 148))SEQ ID NO: 8 PD1 binder CDR3  DKHQSSXYDY (Kabat/Abm) wherein X is W or F(e.g., DKHQSSwYDY (SEQ ID NO: 149) or DKHQSSfYDY) (SEQ ID NO: 150))Name: F023700275 EVQLVESGGGLVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKEREEVAVITWS Description: GGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDKHQSSWYDY 1PD102C12 WGQGTLVTVSS (A14P, A74S, K83R) Target: hPD-1SEQ ID NO: 98 SO 1PD102C12 monomer EVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMGWERQAPGKEREEVA VITWS Name: F023700706 orGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAG DKHQSSWYDY F023700929WGQGTLVTVSS Description:  1PD102C12 (L11V, A14P, A74S, K83R,I89L) Target: hPD-1 SEQ ID NO: 99 Monovalent SODVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKEREEVA VITWS 1PD102C12 +ALB11002 GGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAG DKHQSSWYDYName: F023701127 WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGDescription:  GGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYAD1PD102C12 (E1D, L11V,SVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA A14P, A74S, K83R,I89L)-35GS-ALB11002- A Target: hPD-1 SEQ ID NO: 101 Bivalent SO DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKEREEVA VITWS 1PD102C12 +ALB11002 GGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAG DKHQSSWYDYName: F023700933 WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGDescription: 1PD102C12 GGVVQPGGSLRLSCAAS GSIASIHAMG WFRQAPGKEREFVAVITWSGGITYYAD (E1D, L11V, A14P, A74S,SVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAG DKHQSSWYDY WGQGTLVTK83R, I89L)-35GS- VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGN1PD102C12(L11V, A14P,SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTIA74S, K83R, I89L)-35GS- SRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAALB11002-A Target: hPD-1 SEQ ID NO: 102 Enhanced SO 1PD102C12DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREEVAVITVS monomerGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDY Name: F023701190WGQGTLVTVSS Description: 1PD102C12 (E1D, L11V, A14P, W52aV, A74S,K83R, I89L, W100aF) Target: hPD-1 SEQ ID NO: 103 *Optionally, F023701190comprises an N73X mutation such as N735 Enhanced SO 1PD102C12DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WFRQAPGKEREFVA VITVS monomerGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAG DKHQSSFYDY Name: F023701192WGQGTLVTVSS Description: 1PD102C12 (E1D, L11V, A14P, W52aV, N73Q,A74S, K83R, I89L, W100aF) Target: hPD-1 SEQ ID NO: 104Enhanced SO 1PD102C12 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMGWFRQAPGKEREFVA VITVS monomerGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAG DKHQSSFYDY Name: F023701193WGQGTLVTVSS Description: 1PD102C12 (E1D, L11V, A14P, W52aV,N73P, A74S, K83R, I89L, W100aF) Target: hPD-1 SEQ ID NO: 105 F023700641EVQLVESGGGLVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKEREEVA VITWS Description:GGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAG DKHQSSWYDY1PD102C12 (A14P, A74S,WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG K83R-35GS-GGLVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKEREEVAVITWSGGITYYAD1PD102C12 (A14P, A74S, SVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAG DKHQSSWYDYWGQGTLVT K83R)-35GS-ALB11002VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGN Target: hPD-1SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SEQ ID NO: 126SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 1PD102C12 (E1D, L11V,DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WFRQAPGKEREFVA VITVSA14P, W52aV, N73P, GGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDKHQSSFYDY A74S, K83R, I89L, WGQGTLVTVSS W100aF) amino acids 1-19 of SEQ ID NO: 113 1PD102C12 (E1D, L11V, DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMG WFRQAPGKEREFVA VITVS A14P, W52aV, N73Q,GGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAG DKHQSSFYDYA74S, K83R, I89L, WGQGTLVTVSS W100aF) amino acids 1-19 of SEQ ID NO: 117 1PD102C12 (E1D, L11V, DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMG WFRQAPGKEREFVA VITVS A14P, W52aV, N73S,GGITYYADSVKGRFTISRDSSKNTVYLQMNSLRPEDTALYYCAG DKHQSSFYDYA74S, K83R, I89L, WGQGTLVTVSS W100aF) amino acids 1-19 of SEQ ID NO: 121 *PD1 binder CDRs underscored and/or bold

The present invention includes embodiments wherein a PD1 bindercomprises one, two or three of the CDRs that are in a PD1 binder whichis set forth above in Table A-1 or A-2 (e.g., SEQ ID NO: 57, 98, 99,103, 104 or 105) but which comprises 0, 1, 2, 3, 4, or 5 amino acidsubstitutions, e.g., conservative substitutions, and/or comprises 100,99, 98, 97, 96 or 95% sequence identity relative to the CDR sequencesset forth in Table A-1 or A-2 wherein a PD1 binder having such CDRsretains the ability to bind to PD1. In an embodiment of the invention,the first amino acid of a PD1 binder of the present invention is E. Inan embodiment of the invention, the first amino acid of a PD1 binder ofthe present invention is D. PD1/LAG3 binders comprising such a PD1/LAG3binder are part of the present invention.

The present invention includes any PD1 binder comprising the amino acidsequence of SEQ ID NO: 57, 98, 99, 103, 104 or 105 or an amino acidsequence comprising 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98% or99%) amino acid sequence identity (i.e., comparing the full amino acidsequences) wherein the PD1 binder retains the ability to bind to PD1and, optionally, includes an HSA binder. PD1/LAG3 binders comprisingsuch a PD1/LAG3 binder are part of the present invention.

A PD1 binder described as “102C12 (E1D, L11V, A14P, A74S, K83R, I89L)”refers to a binder having the sequence of 102C12 (SEQ ID NO: 1) orreference A (SEQ ID NO: 2), but wherein the sequence comprises themutations E1D, L11V, A14P, A74S, K83R and I89L. This notation is usedthrough out the specification with regard to several different binders.For example, “11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L)”refers to a LAG3 binder comprising the amino acid sequence of 11B09 (SEQID NO: 64), but wherein the sequence comprises mutations L11V, A14P,R41P, N43K, A62S, A74S, K83R and V89L.

The Kabat residue numbers for certain residues of the PD1 binders setforth in Table A are shown in the sequence below:

(SEQ ID NO: 2) E ₁VQLVESGGGL ₁₁ V ₁₂ Q ₁₃ A ₁₄GGSLRLSCAASG ₂₆ S ₂₇ I ₂₈A ₂₉ S ₃₀IHAMGW ₃₆ F ₃₇ R ₃₈ Q ₃₉AP ₄₁GKERE ₄₆ F ₄₇ V ₄₈ A ₄₉VITW_(52a)SGGITYYADSVKGR ₆₆ F ₆₇ T ₆₈ I ₆₉ S ₇₀RDN ₇₃ A ₇₄KNTVYLQM ₈₂ N_(82a) S _(82b) L _(82c) K ₈₃ P ₈₄EDT ₈₇ A ₈₈ I ₈₉ Y ₉₀ Y ₉₁ CAGDKHQSSW_(100a)YDYW ₁₀₃ G ₁₀₄ Q ₁₀₅ G ₁₀₆ T ₁₀₇ L ₁₀₈ V ₁₀₉ T ₁₁₀ V ₁₁₁ S ₁₁₂ S₁₁₃.

The Kabat residue numbers for certain residues of 102C12 (E1D, L11V,A14P, A74S, K83R, I89L) are shown in the sequence below:

-   D₁VQLVESGGG V₁₁VQP₁₄GGSLRL SCAASGSIAS IHAMGWFRQA PGKEREFVAV    ITWSGGITYY ADSVKGRFTI SRDNS₇₄KNTVY LQMNSLR₈₃PED TAL₈₉YYCAGDK    HQSSWYDYWG QGTLVTVSS (SEQ ID NO: 57). Optionally, residue 1 is an E.

Mutations may be referred to herein and are designated by their Kabatnumber as shown above.

Some preferred, but non-limiting PD1 binders of the invention are 102C12(ElD (optionally), L11V, A14P, A74S, K83R, I89L), comprise the aminoacid sequence set forth in SEQ ID NO: 9-40, 57, 98, 99 or 101-105 or arelisted in FIG. 2, FIG. 3 or FIG. 18(A-P). PD1 binders of SEQ ID NOs: 24to 40, 101 or 102 are examples of PD1 binders of the invention having aC-terminal alanine extension, i.e. an alanine residue at the C-terminalend of the ISVD-sequence (also sometimes referred to as “position 114”)compared to the usual C-terminal sequence VTVSS (SEQ ID NO: 52, aspresent in Reference A). This C-terminal alanine extension can preventthe binding of so-called “pre-existing antibodies” (assumed to be IgGs)to a putative epitope that is situated at the C-terminal region of theISV. This epitope is assumed to include, among other residues, thesurface-exposed amino acid residues of the C-terminal sequence VTVSS(SEQ ID NO: 52) as well as the amino acid residue at position 14 (andthe amino acid residues next/close to the same in the amino acidsequence, such as positions 11, 13 and 15) and may also comprise theamino acid residue at position 83 (and the amino acid residuesnext/close to the same in the amino acid sequence, such as positions 82,82a, 82b and 84) and/or the amino acid residue at position 108 (and theamino acid residues next/close to the same in the amino acid sequence,such as position 107).

However, although the presence of such a C-terminal alanine (or aC-terminal extension generally) can greatly reduce (or, in some cases,essentially fully prevent) the binding of the “pre-existing antibodies”that can be found in the sera from a range of subjects (both healthysubjects and subjects with a medical condition or disease), it has beenfound that the sera from some subjects (such as the sera from patientswith some immune diseases such as SLE) can contain pre-existingantibodies that can bind to the C-terminal region of an ISV (when suchregion is exposed) even when the ISV contains such a C-terminal alanine(or more generally, such C-terminal extension).

Accordingly, one specific objective of the invention is to provide PD1binders that are improved variants of the PD1 Nanobody referred toherein as “Reference A” and that have reduced binding by so-called“pre-existing antibodies”, and in particular of the kind described inPCT/EP2015/060643 (WO2015/173325)(i.e. those pre-existing antibodiesthat can bind to an exposed C-terminal region of an ISV even in thepresence of a C-terminal extension).

The invention provides PD1 binders comprising amino acid sequences thatare variants of the sequence of SEQ ID NO: 1 or 2 which comprise one ormore of the following mutations compared to the sequence of SEQ ID NO: 1or 2:

-   -   1D or 1E;    -   11V;    -   14P;    -   52aV    -   73Q, 73P or 73S    -   74S;    -   83R;    -   89T or 89L;    -   100aF    -   1D or 1E in combination with 11V, 14P, 74S, 83R and 89L;    -   1D in combination with 11V, 14P, 52aV, 73Q 73S or 73P, 74S, 83R,        89L and 100aF;    -   1E in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 89L and 100aF;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R and 100aF, and optionally, 1D;    -   89L in combination with 11V;    -   89L in combination with 110K or 110Q;    -   89L in combination with 112K or 112Q;    -   89L in combination with 11V, 14P,74S, 83R, and, optionally, 1D;    -   110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L and 100aF and optionally, 1D;    -   112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L and 100aF and optionally, 1D;    -   89L in combination with 11V and 110K or 110Q;    -   89L in combination with 11V and 112K or 112Q;    -   11V in combination with 110K or 110Q; or    -   11V in combination with 112K or 112Q.

In particular, the PD1 binders (e.g., an ISVD such as a Nanobody)provided by the invention comprise a variant of SEQ ID NO: 1 or 2wherein, in an embodiment of the invention:

-   -   the amino acid residue at position 1 is selected from E and D;    -   the amino acid residue at position 11 is selected from L and V;    -   the amino acid residue at position 14 is selected from A and P;    -   the amino acid residue at position 52a is selected from W and V;    -   the amino acid residue at position 73 is selected from N, S, P        and Q;    -   the amino acid residue at position 74 is selected from A or S;    -   the amino acid residue at position 83 is selected from K or R;    -   the amino acid residue at position 89 is selected from T, V, I        or L;    -   the amino acid residue at position 100a is selected from W and        F;    -   the amino acid residue at position 110 is selected from T, K or        Q; and/or    -   the amino acid residue at position 112 is selected from S, K or        Q;        for example, wherein the PD1 binder comprises one or more of the        following mutations:    -   (i) position 1 is D or E;    -   (ii) position 11 is V;    -   (iii) position 14 is P;    -   (iv) position 52a is V;    -   (v) position 73 is P, S or Q;    -   (vi) position 74 is S;    -   (vii) position 83 is R;    -   (viii) position 89 is T or L;    -   (ix) position 100a is F;        for example, comprising a set of mutations as follows:    -   a. position 1 is D or E, position 11 is V, position 14 is P,        position 74 is S, position 83 is R; and position 89 is L;    -   b. position 1 is D or E, position 11 is V, position 14 is P,        position 52a is V; position 73 is S, P or Q; position 74 is S,        position 83 is R; position 89 is L; and position 100a is F;    -   c. position 89 is L and position 11 is V;    -   d. position 89 is L and position 110 is K or Q;    -   e. position 89 is L and position 112 is K or Q;    -   f. position 1 is D or E, position 11 is V, position 14 is P,        position 52a is V; position 73 is S, P or Q; position 74 is S,        position 83 is R; position 89 is L; position 100a is F and        position 110 is K or Q;    -   g. position 1 is D or E, position 11 is V, position 14 is P,        position 52a is V; position 73 is S, P or Q; position 74 is S,        position 83 is R; position 89 is L; position 100a is F and        position 112 is K or Q;    -   h. position 89 is L and position 11 is V and position 110 is K        or Q;    -   i. position 89 is L and position 11 is V and position 112 is K        or Q;    -   j. position 11 is V and position 110 is K or Q; and/or    -   k. position 11 is V and position 112 is K or Q;        relative to the amino acid sequence of SEQ ID NO: 1 or 2

In particular embodiments, the PD1 binders (e.g., an ISVD such as aNanobody) of the invention comprise amino acid sequences that arevariants of SEQ ID NO: 1 or SEQ ID NO: 2 in which position 89 is T or L,or in which 1 is D or E, 11 is V, 14 is P, 74 is S, 83 is R and 89 is Lor in which position 11 is V and position 89 is L (optionally insuitable combination with a 110K or 110Q mutation and/or a 112K or 112Qmutation, and in particular in combination with a 110K or 110Q mutation)are also part of the present invention. The present invention includesamino acid sequences in which position 11 is V and position 89 is L,optionally with a 110K or 110Q mutation.

As mentioned, the PD1 binders provided by the invention described hereincan bind (and in particular, can specifically bind) to PD-1. In anembodiment of the invention, they can bind to PD1 and inhibit bindingbetween PD1 and PD-L1 and/or PD-L2. For example, in an embodiment of theinvention, the PD1 binders of the present invention,

binds to PD-1 and releases T-cells from PD-1 pathway—mediated inhibitionof the T-cell mediated immune response (e.g., by releasing the T-cellsfrom PD1 mediated inhibition of proliferation and cytokine production)

Table B lists some non-limiting possible combinations of the amino acidresidues that can be present at positions 11, 89, 110 and 112 in the PD1binders of the invention.

TABLE B Possible Combinations of Mutations at Amino Acid Positions 11,89, 110 and 112 in PD1 Binder Variants of SEQ ID NOs: 1 or 2. POSITIONPOSITION 11 89 110 112 11 89 110 112 COM- L T T S COM- V T T S BINA- L TT K BINA- V T T K TION L T T Q TION V T T Q L T K S V T K S L T Q S V TQ S L V T K V V T K L V T Q V V T Q L V K S V V K S L V Q S V V Q S L IT K V I T K L I T Q V I T Q L I K S V I K S L I Q S V I Q S V L T S L LT K V L T K L L T Q V L T Q L L K S V L K S L L Q S V L Q S Thesepositions may be combined with the mutations such as E1D, A14P, A74Sand/or K83R (and/or others).

The PD1 binders provided by the invention are further as described inthe description, examples and figures herein, i.e. they have CDRs thatare as described herein and have an overall degree of sequence identity(as defined herein) with the sequence of SEQ ID NO: 1 or 2 that is asdisclosed herein and/or may have a limited number of “amino aciddifferences” (as described herein) with (one of) these referencesequences.

The PD1 binders (e.g., ISVDs such as Nanobodies) of the invention thatcomprise the amino acid sequence of SEQ ID NO:1 or 2 and one or moremutations at position 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110 and/or112 preferably include the following CDRs (according to the Kabatconvention):

-   -   a CDR1 (according to Kabat) that is the amino acid sequence        IHAMG (SEQ ID NO: 3); and    -   a CDR2 (according to Kabat) that is the amino acid sequence        VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V); and    -   a CDR3 (according to Kabat) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F); optionally,        wherein CDR1, CDR2 and/or CDR3 has 1, 2, 3, 4, 5, 6, 7, 8, 9 or        10 substitutions, e.g., conservative substitutions.

Alternatively, when the CDRs are given according to the Abm convention,the PD1 binders (e.g., ISVDs such as Nanobodies) of the inventionpreferably comprise the following CDRs:

-   -   a CDR1 (according to Abm) that is the amino acid sequence        GSIASIHAMG (SEQ ID NO: 6); and    -   a CDR2 (according to Abm) that is the amino acid sequence        VITXSGGITY (SEQ ID NO: 7, wherein X is W or V); and    -   a CDR3 (according to Abm) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 8, which is the same as SEQ ID NO: 5;        wherein X is W or F); optionally, wherein CDR1, CDR2 and/or CDR3        has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, e.g.,        conservative substitutions.

A PD1 binder (e.g., ISVDs such as Nanobodies) of the invention, inaddition to the CDR1, CDR2 and CDR3 set forth above, preferably alsohas:

-   -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 of at least 85%, preferably at least 90%, more        preferably at least 95% (e.g., 100%) (in which the CDRs, any        C-terminal extension that may be present, as well as the        mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110        and/or 112 required by the specific aspect involved are not        taken into account for determining the degree of sequence        identity) when the comparison is performed by a BLAST algorithm        wherein the parameters of the algorithm are selected to give the        largest match between the respective sequences over the entire        length of the respective reference sequences (e.g., expect        threshold: 10; word size: 3; max matches in a query range: 0;        BLOSUM 62 matrix; gap costs: existence 11, extension 1;        conditional compositional score matrix adjustment); and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs; not taking into account any        C-terminal extension that may be present and not taking into        account the mutations at positions 1, 11, 14, 52a, 73, 74, 83,        89, 100a, 110 and/or 112 required by the specific aspect        involved).

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J.272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol.215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden,T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., etal., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997)Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem.17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model ofevolutionary change in proteins.” in Atlas of Protein Sequence andStructure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352,Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al.,“Matrices for detecting distant relationships.” in Atlas of ProteinSequence and Structure, (1978) vol. 5, suppl. 3.” M.O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991)Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York.

With regards to the various aspects and preferred aspects of the PD1binders (e.g., ISVD such as a Nanobody) of the invention provided by theinvention, when it comes to the degree of overall sequence identity withrespect to SEQ ID NO: 1 or 2 and/or the number and kind of “amino aciddifferences” that may be present in such a binder of the invention (i.e.compared to the sequence of SEQ ID NO: 1 or 2), it should be noted that,unless otherwise stated, when it is said that

-   -   (i) an amino acid sequence of the invention has a degree of        sequence identity with the sequence of SEQ ID NO: 1 or 2 of at        least 85%, preferably at least 90%, more preferably at least 95%        (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity); and/or when it is said that    -   (ii) an amino acid sequence of the invention has no more than 7,        preferably no more than 5, such as only 3, 2 or 1 “amino acid        differences” with the sequence of SEQ ID NO: 1 or 2 (again, not        taking into account any C-terminal extension that may be present        and not taking into account the mutations at positions 1, 11,        14, 52a, 73, 74, 83, 89, 100a, 110 and/or 112 required by the        specific aspect involved),        then this also includes sequences that have no amino acid        differences with the sequence of SEQ ID NO: 1 or 2 other than        the mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a,        110 and/or 112 (as required by the specific aspect involved) and        any C-terminal extension that may be present.

Thus, in one specific aspect of the invention, the PD1 binders of theinvention comprising the amino acid sequence of SEQ ID NO: 1 or 2, butwith at least one amino acid mutation at positon 1, 11, 14, 52a, 73, 74,83, 89, 100a, 110 and/or 112, may have 100% sequence identity with SEQID NO: 1 or 2 (including a CDR1, CDR2 and CDR3 thereof optionallycomprising a W52aV and/or W100aF mutation, but not taking into accountthe mutation(s) or combination of mutations at positions 1, 11, 14, 52a,73, 74, 83, 89, 100a, 110 and/or 112 disclosed herein and/or anyC-terminal extension that may be present) and/or may have no amino aciddifferences with SEQ ID NO: 1 or 2 (i.e. other than the mutation(s) orcombination of mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89,100a, 110 and/or 112 disclosed herein and any C-terminal extension thatmay be present).

When any amino acid differences are present (i.e. besides any C-terminalextension and the mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89,100a, 110 and/or 112 that are required by the specific aspect of theinvention involved), these amino acid differences may be present in the

CDRs and/or in the framework regions, but they are preferably presentonly in the framework regions (as defined by the Abm convention, i.e.not in the CDRs as defined according to the Abm convention), i.e. suchthat the PD1 binders of the invention have the same CDRs (definedaccording to the Abm convention) as are present in SEQ ID NO: 1, 2,9-40, 57, 98, 99, 101, 102, 103, 104 or 105.

Also, when a PD1 binder of the invention according to any aspect of theinvention has one or more amino acid differences with the sequence ofSEQ ID NO: 1 or 2 (besides the mutations at positions 1, 11, 14, 52a,73, 74, 83, 89, 100a, 110 and/or 112 that are required by the specificaspect involved), then some specific, but non-limiting examples of suchmutations/amino acid differences that may be present (i.e. compared tothe sequences of SEQ ID NO: 1 or 2) are for example “humanizing”substitutions; reference is for example made to WO 09/138519 (or in theprior art cited in WO 09/138519) and WO 08/020079 (or in the prior artcited in WO 08/020079), as well as Tables A-3 to A-8 from WO 08/020079(which are lists showing possible humanizing substitutions).

Also, when the PD-1 binders of the invention are present at and/or formthe N-terminal part of the polypeptide in which they are present, thenthey preferably contain a D at position 1 (i.e. an E1D mutation comparedto Reference A). A preferred but non-limiting example of such anN-terminal PD-1 binder is given as SEQ ID NOs: 24 or 57. Accordingly, ina further aspect, the invention relates to a polypeptide (which is asfurther described herein) that has a PD-1 binder (which is as furtherdescribed herein) at its N-terminal end, wherein said PD-1 binder has aD at position 1, and is for example SEQ ID NOs: 24, 25, 57 or 101-105.

Similarly, when a PD-1 binder of the invention is used in monovalentformat, it preferably has both a C-terminal extension X(n) as describedherein and a D at position 1. A preferred but non-limiting example ofsuch a monovalent PD-1 binder is given as SEQ ID NO: 40. Accordingly, ina further aspect, the invention relates to a monovalent PD-1 binder ofthe invention (which is as further described herein) that has a D atposition 1 and a C-terminal extension X(n) (which is preferably a singleAla residue). In one specific aspect, said monovalent PD-1 binder is SEQID NO: 40, 101 or 102.

By means of preferred, but non-limiting examples, SEQ ID NOs: 23, 24,39, 40 and 57 are examples of PD-1 binders of the invention having aminoacid differences with SEQ ID NO: 1 or 2 such as A14P, A74S and/or K83R(in addition, as indicated in the previous paragraphs, SEQ ID NOs: 24and 40 and 57 also have a ElD mutation). Thus, in a specific aspect, theinvention relates to PD-1 binders of the invention (i.e. havingmutations at positions 11, 89, 110 and/or 112 as described herein andalso further being as described herein) that at least have a suitablecombination of an optional ElD mutation, an A14P mutation, an A74Smutation, a K83R mutation and/or I89L mutation and, preferably, asuitable combination of any two of these mutations, such as all of thesemutations.

The PD1 binders of the invention, when they are used in a monovalentformat and/or when they are present at and/or form the C-terminal end ofthe polypeptide in which they are present (or when they otherwise havean “exposed” C-terminal end in such polypeptide by which is generallymeant that the C-terminal end of the ISVD is not associated with orlinked to a constant domain (such as a CHI domain)); (see WO 12/175741and PCT/EP2015/060643 (WO2015/173325)), preferably also have aC-terminal extension of the formula (X)_(n), in which n is 1 to 10,preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, suchas 1); and each X is an (preferably naturally occurring) amino acidresidue that is independently chosen from naturally occurring amino acidresidues (although according to preferred one aspect, it does notcomprise any cysteine residues), and preferably independently chosenfrom the group consisting of alanine (A), glycine (G), valine (V),leucine (L) or isoleucine (I).

According to some preferred, but non-limiting examples of suchC-terminal extensions X_((n)), X and n can be as follows:

-   (a) n=1 and X=Ala;-   (b) n=2 and each X=Ala;-   (c) n=3 and each X=Ala;-   (d) n=2 and at least one X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (e) n=3 and at least one X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (f) n=3 and at least two X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (g) n=1 and X=Gly;-   (h) n=2 and each X=Gly;-   (i) n=3 and each X=Gly;-   (j) n=2 and at least one X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (k) n=3 and at least one X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (l) n=3 and at least two X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (m) n=2 and each X=Ala or Gly;-   (n) n=3 and each X=Ala or Gly;-   (o) n=3 and at least one X=Ala or Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile); or-   (p) n=3 and at least two X=Ala or Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);    with aspects (a), (b), (c), (g), (h), (i), (m) and (n) being    particularly preferred, with aspects in which n=1 or 2 being    preferred and aspects in which n=1 being particularly preferred.

It should also be noted that, preferably, any C-terminal extensionpresent in a PD1 binder of the invention does not contain a (free)cysteine residue (unless said cysteine residue is used or intended forfurther functionalization, for example for PEGylation).

Some specific, but non-limiting examples of useful C-terminal extensionsare the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA,AAG, AGG, AGA, GGA, GAA or GAG.

When the PD1 binders of the invention contain mutations at positions 110or 112 (optionally in combination with mutations at position 1, 11, 14,52a, 73, 74, 83, 89 and/or 100a as described herein), the C-terminalamino acid residues of framework 4 (starting from position 109) can, inan embodiment of the invention be as set forth in SEQ ID NO: 1, 2, 9-40,57, 100, 101, 103, 104, 105, 106 or 107 but wherein the 5 C-terminalresidues can be substituted as follows:

-   -   (i) if no C-terminal extension is present: VTVKS (SEQ ID NO:        42), VTVQS (SEQ ID NO: 43), VKVSS (SEQ ID NO: 44) or VQVSS (SEQ        ID NO: 45); or    -   (ii) if a C-terminal extension is present: VTVKSX_((n)) (SEQ ID        NO: 46), VTVQSX(n) (SEQ ID NO: 47), VKVSSX(n) (SEQ ID NO: 48) or        VQVSSX₍n₎ (SEQ ID NO: 49), such as VTVKSA (SEQ ID NO: 50),        VTVQSA (SEQ ID NO: 51), VKVSSA (SEQ ID NO: 52) or VQVSSA (SEQ ID        NO: 53).

When the PD1 binders of the invention contain or, alternatively, do notcontain mutations at positions 110 or 112 (but only mutations atposition 1, 11, 14, 52a, 73, 74, 83, 89 and/or 100a as describedherein), the C-terminal amino acid residues of framework 4 (startingfrom position 109) can, in an embodiment of the invention be as setforth in SEQ ID NO: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104 or 105but wherein the 5C-terminal residues can be substituted as follows:

-   -   when no C-terminal extension is present: VTVSS (SEQ ID NO: 54)        (as in the sequence of SEQ ID NO: 1 or 2); or    -   (ii) when a C-terminal extension is present: VTVSSX_((n)) (SEQ        ID NO: 55) such as VTVSSA (SEQ ID NO: 56). In these C-terminal        sequences, X and n are as defined herein for the C-terminal        extensions.

Some preferred but non-limiting examples of PD1 binders of the inventionare given in SEQ ID NOs: 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 andeach of these sequences forms a further aspect of the invention. Ofthese, the PD1 binders of SEQ ID NOs: 9-24, 57, 98, 99, 103, 104 and 105do not have a C-terminal extension, and the PD1 binders of SEQ ID NOs:25-40, 101 and 102 contain a C-terminal alanine (which is a preferredbut non-limiting example of a C-terminal extension as described herein).

Examples of PD1 binders of the present invention comprise the amino acidsequence set forth in SEQ ID NO: 23, 24, 39, 40, 57, 98, 99, 101, 102,103, 104 or 105.

Thus, in an embodiment of the invention, a PD1 binder (e.g., an ISVDsuch as a Nanobody) comprises:

-   -   a CDR1 (according to Kabat) that is the amino acid sequence        IHAMG (SEQ ID NO: 3); and    -   a CDR2 (according to Kabat) that is the amino acid sequence        VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V); and    -   a CDR3 (according to Kabat) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and also has:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 of at least 85%, preferably at least 90%, more        preferably at least 95% (e.g., 100%) (in which the CDRs thereof,        optionally comprising a W52aV and/or W100aF mutation, any        C-terminal extension that may be present, as well as the        mutations at positions 1, 11, 14, 52a, 73, 74, 83, 89, 100a, 110        and/or 112 required by the specific aspect involved are not        taken into account for determining the degree of sequence        identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally has:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        in which, in an embodiment of the invention:    -   the amino acid residue at position 1 is E or D;    -   the amino acid residue at position 11 is L or V;    -   the amino acid residue at position 14 is A or P;    -   the amino acid residue at position 52a is W or V;    -   the amino acid residue at position 73 is N, S, P or Q;    -   the amino acid residue at position 74 is A or S;    -   the amino acid residue at position 83 is K or R;    -   the amino acid residue at position 89 is T, V, I or L;    -   the amino acid residue at position 100a is W or F;    -   the amino acid residue at position 110 is T, K or Q; and    -   the amino acid residue at position 112 is S, K or Q;        for example, wherein the PD1 binder comprises one or more of the        following mutations:    -   (i) position 1 is E or D;    -   (ii) position 11 is V;    -   (iii) position 14 is P;    -   (iv) position 52a is V;    -   (v) position 73 is S, P or Q;    -   (vi) position 74 is S;    -   (vii) position 83 is R;    -   (viii) position 89 is L;    -   (ix) position 100a is F;        for example, comprising a set of mutations as follows:    -   a. position 1 is D or E, position 11 is V, position 14 is P,        position 74 is S, position 83 is R and position 89 is L;    -   b. position 1 is D or E, position 11 is V, position 14 is P,        position 52a is V; position 73 is S, P or Q; position 74 is S,        position 83 is R, position 89 is L and position 100a is F;    -   c. position 1 is D or E, position 11 is V, position 14 is P,        position 74 is S, position 83 is R and position 89 is L;    -   d. position 89 is L and position 11 is V;    -   e. position 89 is L and position 110 is K or Q;    -   f. position 89 is L and position 112 is K or Q;    -   g. position 89 is L and position 11 is V and position 110 is K        or Q;    -   h. position 89 is L and position 11 is V and position 112 is K        or Q;    -   i. 110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q        or 73P, 74S, 83R, 89L and 100aF, and optionally, 1D or 1E; or in        combination with 11V, 14P, 74S, 83R, 89L and optionally, 1D or        1E;    -   j. 112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q        or 73P, 74S, 83R, 89L and 100a F and, optionally, 1D or 1E; or        in combination with 11V, 14P, 74S, 83R, 89L and optionally, 1D        or 1E;    -   k. position 11 is V and position 110 is K or Q; or    -   l. position 11 is V and position 112 is K or Q.

In a further aspect, the invention relates to a PD1 binder (e.g., anISVD such as a Nanobody) having:

-   -   a CDR1 (according to Kabat) that is the amino acid sequence        IHAMG (SEQ ID NO: 3); and    -   a CDR2 (according to Kabat) that is the amino acid sequence        VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V); and    -   a CDR3 (according to Kabat) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and also having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be        present as well as the CDRs thereof, optionally comprising a        W52aV and/or W100aF mutation, are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (e.g.,        100%) (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        which PD1 binder (e.g., an ISVD such as a Nanobody) comprises,        in an embodiment of the invention, one or more of the following        amino acid residues (i.e. mutations compared to the amino acid        sequence of SEQ ID NO: 1 or 2) at the positions mentioned        (numbering according to Kabat):    -   1D or 1E;    -   11V;    -   14P;    -   52aV;    -   73Q, 73P or 73S;    -   74S;    -   83R;    -   89T or 89L; or    -   100aF;        for example, comprising a set of mutations as follows:    -   1D or 1E in combination with 11V, 14P, 52aV, 73S or 73Q or 73P,        74S, 83R, 89L and/or 100aF;    -   1D or 1E in combination with 11V, 14P, 74S, 83R and/or 89L;    -   89L in combination with 11V;    -   89L in combination with 110K or 110Q;    -   89L in combination with 112K or 112Q;    -   89L in combination with 11V, 14P,74S, 83R and 1D and 1E;    -   110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L and/or 100aF and 1D or 1E;    -   112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L and/or 100aF and 1D or 1E;    -   110K or 110Q in combination with 11V, 14P, 74S, 83R, 89L and/or        1D or 1E;    -   112K or 112Q in combination with 11V, 14P, 74S, 83R, 89L and/or        1D or 1E;    -   89L in combination with 11V and 110K or 110Q;    -   89L in combination with 11V and 112K or 112Q;    -   11V in combination with 110K or 110Q; or    -   11V in combination with 112K or 112Q.

As mentioned, when a PD1 binder (e.g., an ISVD such as a Nanobody) ofthe invention is used in a monovalent format and/or is present at theC-terminal end of a polypeptide (as defined herein), the PD1 binderpreferably has a C-terminal extension X(n), which C-terminal extensionmay be as described herein for the PD1 binders of the invention and/oras described in WO 12/175741 or PCT/EP2015/060643 (WO2015/173325).

Some preferred, but non-limiting examples of PD1 binders (e.g., an ISVDsuch as a Nanobody) of the invention are given in SEQ ID NOs: 9-40, 57,98, 99, 101, 102, 103, 104 and 105 and each of these amino acidsequences individually forms a further aspect of the invention.

As mentioned, the invention includes PD1 binders comprising amino acidsequences of SEQ ID NO: 1 or 2 but wherein position 89 is T; or in whichposition 1 is E or D, position 11 is V, position 14 is P, position 52ais V, position 73 is P, S or Q, position 74 is S, position 83 is R,position 89 is L and/or positon 100a is F; or in which position 11 is Vand position 89 is L (optionally in suitable combination with a 110K or110Q mutation and/or a 112K or 112Q mutation, and in particular incombination with a 110K or 110Q mutation). In an embodiment of theinvention, the amino acid sequences in which position 11 is V andposition 89 is L, optionally with a 110K or 110Q mutation.

Thus, in one preferred aspect, the invention relates to a PD1 binder(e.g., an immunoglobulin single variable domain (ISVD) such as aNanobody) having:

-   -   a CDR1 (according to Kabat) that is the amino acid sequence        IHAMG (SEQ ID NO: 3); and    -   a CDR2 (according to Kabat) that is the amino acid sequence        VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V); and    -   a CDR3 (according to Kabat) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and also having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be        present as well as the CDRs thereof, optionally comprising a        W52aV and/or W100aF mutation, are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (e.g.,        100%) (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        wherein, in an embodiment of the invention:    -   the amino acid at position 1 is E or D;    -   the amino acid at position 11 is L or V;    -   the amino acid at position 14 is A or P;    -   the amino acid at position 52a is W or V;    -   the amino acid at position 73 is N, S, P or Q;    -   the amino acid at position 74 is A or S;    -   the amino acid at position 83 is K or R;    -   the amino acid at position 89 is I, T or L;    -   the amino acid at position 100a is W or F;    -   the amino acid residue at position 110 is T, K or Q (and is        preferably T); and    -   the amino acid residue at position 112 is S, K or Q (and is        preferably S).

In another preferred aspect, the invention relates to a PD1 binder(e.g., an immunoglobulin single variable domain (ISVD) such as aNanobody) having:

-   -   a CDR1 (according to Kabat) that is the amino acid sequence        IHAMG (SEQ ID NO: 3); and    -   a CDR2 (according to Kabat) that is the amino acid sequence        VITXSGGITYYADSVKG (SEQ ID NO: 4; wherein X is W or V); and    -   a CDR3 (according to Kabat) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be        present as well as the CDRs thereof, optionally comprising a        W52aV and/or W100aF mutation, are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (e.g.,        100%) (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        in which one or more of the following is true:    -   the amino acid at position 1 is E or D;    -   the amino acid at position 11 is L or V;    -   the amino acid at position 14 is A or P;    -   the amino acid at position 52a is W or V;    -   the amino acid at position 73 is S, P, N or Q;    -   the amino acid at position 74 is A or S;    -   the amino acid at position 83 is K or R;    -   the amino acid at position 89 is I, T or L;    -   the amino acid at position 100a is W or F;    -   the amino acid residue at position 110 is T, K or Q (and is        preferably T); or    -   the amino acid residue at position 112 is S, K or Q (and is        preferably S).

In one specific, but non-limiting aspect, the PD1 binders (e.g., an ISVDsuch as a Nanobody) of the invention comprise one or more of thefollowing amino acid residues (i.e. mutations compared to the sequenceof SEQ ID NO: 1 or 2) at the positions mentioned (numbering according toKabat):

-   -   11V in combination with 89L;    -   11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R,        89L and/or 100aF and, optionally, 1D;    -   11V in combination with 14P, 74S, 83R, 89L and 1D or 1E;    -   11V in combination with 110K or 110Q;    -   11V in combination with 112K or 112Q;    -   11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R,        89L, 100aF, 110K or 110Q and/or 1D or 1E;    -   11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R,        89L, 100aF, 112K or 112Q and/or 1D or 1E;    -   11V in combination with 89L and 110K or 110Q;    -   11V in combination with 89L and 112K or 112Q; or    -   11V in combination with 1D or 1E, 14P, 52aV, 73S or 73Q or 73P,        74S, 83R, 89L and/or 100aF;        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 9-40, 57, 98, 99, 101, 102, 103, 104        or 105 (e.g., according to Kabat) and have an overall degree of        sequence identity with the amino acid sequence of SEQ ID NO: 1        or 2 that are as described herein.

In another specific, but non-limiting aspect, the PD1 binders (e.g., anISVD such as a Nanobody) of the invention comprise one or more of thefollowing amino acid residues (i.e. mutations compared to the sequenceof SEQ ID NO: 1 or 2) at the positions mentioned (numbering according toKabat):

-   -   89L in combination with 11V;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF and/or 1D or 1E;    -   89L in combination with 11V, 14P, 74S, 83R, and/or 1D or 1E;    -   89L in combination with 110K or 110Q;    -   89L in combination with 112K or 112Q;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF, 110K or 110Q and/or 1D or 1E;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF, 112K or 112Q and/or 1D or 1E;    -   89L in combination with 11V, 14P, 74S, 83R, 110K or 110Q and/or        1D or 1E;    -   89L in combination with 11V, 14P, 74S, 83R, 112K or 112Q and/or        1D or 1E;    -   89L in combination with 11V and 110K or 110Q; or    -   89L in combination with 11V and 112K or 112Q;        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 9-40, 57, 98, 99, 101, 102, 103, 104        or 105 (e.g., according to Kabat) and have an overall degree of        sequence identity with the amino acid sequence of SEQ ID NO: 1        or 2 that are as described herein.

In another specific, but non-limiting aspect, the PD1 binders (e.g., anISVD such as a Nanobody) of the invention comprise one or more of thefollowing amino acid residues (i.e. mutations compared to the sequenceof SEQ ID NO: 1 or 2) at the positions mentioned (numbering according toKabat):

-   -   110K or 110Q in combination with 1D;    -   110K or 110Q in combination with 1E;    -   110K or 110Q in combination with 11V;    -   110K or 110Q in combination with 14P;    -   110K or 110Q in combination with 52aV;    -   110K or 110Q in combination with 73S or 73Q or 73P;    -   110K or 110Q in combination with 74S;    -   110K or 110Q in combination with 83R;    -   110K or 110Q in combination with 89L;    -   110K or 110Q in combination with 100aF;    -   110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L, 100aF and/or 1D or 1E;    -   110K or 110Q in combination with 11V, 14P, 74S, 83R, 89L and/or        1D or 1E;    -   110K or 110Q in combination with 89L; or    -   110K or 110Q in combination with 11V and 89L;        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 9-40, 57, 98, 99, 101, 102, 103, 104        or 105 (e.g., according to Kabat) and have an overall degree of        sequence identity with the amino acid sequence of SEQ ID NO: 1        or 2 that are as described herein.

In another specific, but non-limiting aspect, the PD1 binders (e.g., anISVD such as a Nanobody) of the invention comprise one or more of thefollowing amino acid residues (i.e. mutations compared to the sequenceof SEQ ID NO: 1 or 2) at the positions mentioned (numbering according toKabat):

-   -   112K or 112Q in combination with 1D or 1E;    -   112K or 112Q in combination with 11V;    -   112K or 112Q in combination with 14P;    -   112K or 112Q in combination with 52aV;    -   112K or 112Q in combination with 73S or 73Q or 73P;    -   112K or 112Q in combination with 74S;    -   112K or 112Q in combination with 83R;    -   112K or 112Q in combination with 89L;    -   112K or 112Q in combination with 100aF;    -   112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L, 100aF and/or 1D or 1E;    -   112K or 112Q in combination with 11V, 14P, 74S, 83R, 89L and/or        1D or 1E; or    -   112K or 112Q in combination with 11V and 89L;        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 9-40, 57, 98, 99, 101, 102, 103, 104        or 105 (e.g., according to Kabat) and have an overall degree of        sequence identity with the amino acid sequence of SEQ ID NO: 1        or 2 that are as described herein.

In another aspect, the PD1 binders (e.g., an ISVD such as a Nanobody) ofthe invention comprise a T at position 89 and have CDRs that are thesame as the CDRs that are present in the sequence of SEQ ID NO: 9-40,57, 98, 99, 101, 102, 103, 104 or 105 (e.g., according to Kabat) andhave an overall degree of sequence identity with the amino acid sequenceof SEQ ID NO: 1 or 2 that are as described herein.

In another aspect, the PD1 binders (e.g., an ISVD such as a Nanobody) ofthe invention comprise a V at position 11 and an L at position 89 andhave CDRs that are the same as the CDRs that are present in the sequenceof SEQ ID NO: 9-40, 57, 100, 101, 102 103, 104 or 105 (e.g., accordingto Kabat) and have an overall degree of sequence identity with the aminoacid sequence of SEQ ID NO: 1 or 2 that are as described herein.

As mentioned, the PD1 binders (e.g., an ISVD such as a Nanobody) of theinvention according to the above aspects are preferably further suchthat they contain a suitable combination of an A14P mutation, an A74Smutation and/or a K83R mutation, and preferably a suitable combinationof any two of these mutations, such as all three of these mutations(e.g., E1D (optionally), L11V, A14P, W52aV, N73S or N73Q or N73P, A74S,K83R, I89L and W100aF). When a LAG3 or HSA binder is present at theN-terminal end of a PD1 binder of the present invention, the N-terminalbinding moiety preferably has an E1D mutation.

In another aspect, the invention relates to a PD1 binder (e.g., animmunoglobulin single variable domain (ISVD) such as a Nanobody) having:

-   -   a CDR1 (according to Abm) that is the amino acid sequence        GSIASIHAMG (SEQ ID NO: 6); and    -   a CDR2 (according to Abm) that is the amino acid sequence        VITXSGGITY (SEQ ID NO: 7, wherein X is W or V); and    -   a CDR3 (according to Abm) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and also having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be        present as well as the CDRs, optionally comprising a W52aV        and/or W100aF mutation, are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (e.g.,        100%) (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        wherein, in an embodiment of the invention:    -   the amino acid at position 1 is E or D;    -   the amino acid at position 11 is L or V;    -   the amino acid at position 14 is A or P;    -   the amino acid at position 52a is W or V;    -   the amino acid at position 73 is N, S, P or Q;    -   the amino acid at position 74 is A or S;    -   the amino acid at position 83 is K or R;    -   the amino acid at position 89 is V, I, T or L;    -   the amino acid residue at position 89 is T;    -   the amino acid at position 100a is W and F;    -   the amino acid residue at position 110 is T, K or Q (and is        preferably T); and    -   the amino acid residue at position 112 is S, K or Q (and is        preferably S)        for example, in an embodiment of the invention, the PD1 binder        comprises one or more of the following mutations:

-   (i) position 1 is D or E;

-   (ii) position 11 is V;

-   (iii) position 14 is P;

-   (iv) position 52a is V;

-   (v) position 73 is Q, P or S;

-   (vi) position 74 is S;

-   (vii) position 83 is R;

-   (viii) position 89 is L or T;

-   (ix) position 100a is F;    for example, comprising a set of mutations as follows:

-   a. position 11 is V; position 14 is P; position 52a is V; position    73 is P, S or Q; position 74 is S; position 83 is R; position 89 is    L; position 100a is F; and, optionally, position 1 is E or D;

-   b. position 11 is V; position 14 is P; position 74 is S; position 83    is R; position 89 is L; and, optionally, position 1 is E or D;

-   c. position 89 is L and position 11 is V;

-   d. position 89 is L and position 110 is K or Q;

-   e. position 89 is L and position 112 is K or Q;

-   f. position 11 is V; position 14 is P; position 52a is V; position    73 is P, S or Q; position 74 is S; position 83 is R; position 89 is    L; position 100a is F; position 110 is K or Q and, optionally,    position 1 is E or D;

-   g. position 11 is V; position 14 is P; position 52a is V; position    73 is S, P or Q; position 74 is S; position 83 is R; position 89 is    L; position 100a is F; position 112 is K or Q and, optionally,    position 1 is E or D;

-   h. position 11 is V; position 14 is P; position 74 is S; position 83    is R; position 89 is L; position 110 is K or Q and, optionally,    position 1 is E or D;

-   i. position 11 is V; position 14 is P; position 74 is S; position 83    is R; position 89 is L; position 112 is K or Q and, optionally,    position 1 is E or D;

-   j. position 89 is L and position 11 is V and position 110 is K or Q;

-   k. position 89 is L and position 11 is V and position 112 is K or Q;

-   l. position 11 is V and position 110 is K or Q; or

-   m. position 11 is V and position 112 is K or Q.

In a further aspect, the invention relates to a PD1 binder (e.g., animmunoglobulin single variable domain (ISVD) such as a Nanobody) having:

-   -   a CDR1 (according to Abm) that is the amino acid sequence        GSIASIHANIG (SEQ ID NO: 6); and    -   a CDR2 (according to Abm) that is the amino acid sequence        VITXSGGITY (SEQ ID NO: 7, wherein X is W or V); and    -   a CDR3 (according to Abm) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and also having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be        present as well as the CDRs, optionally comprising a W52aV        and/or W100aF mutation, are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (e.g.,        100%) (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        which immunoglobulin single variable domain comprises one or        more of the following amino acid residues (i.e. mutations        compared to the amino acid sequence of SEQ ID NO: 1 or 2) at the        positions mentioned (numbering according to Kabat):    -   1D or 1E;    -   11V;    -   14P;    -   52aV;    -   73Q, 73S or 73P;    -   74S;    -   83R;    -   89L or 89T; or    -   100aF;        for example, comprising a set of mutations as follows:    -   89T or 89L in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 100aF or 1E or 1D;    -   89T or 89L in combination with 11V, 14P, 74S, 83R, or 1E or 1D;    -   89L in combination with 11V;    -   89L in combination with 110K or 110Q;    -   89L in combination with 112K or 112Q;    -   89L in combination with 11V, 14P, 74S, 83R, 110K or 110Q or 1E        or 1D;    -   89L in combination with 11V, 14P, 74S, 83R, 112K or 112Q or 1E        or 1D;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF, 110K or 110Q or lE or 1D;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF, 112K or 112Q or lE or 1D;    -   89L in combination with 11V and 110K or 110Q;    -   89L in combination with 11V and 112K or 112Q;    -   11V in combination with 110K or 110Q; or    -   11V in combination with 112K or 112Q.

As mentioned, when a PD1 binder (e.g., an ISVD such as a Nanobody) ofthe invention is used in a monovalent format and/or wherein the moietythat binds to PD1 is present at the C-terminal end of a polypeptide (asdefined herein), the PD1 binding moiety preferably has a C-terminalextension X(n), which C-terminal extension may be as described hereinfor the PD1 binders of the invention and/or as described in WO 12/175741or PCT/EP2015/060643 (WO2015/173325).

Some preferred, but non-limiting examples of PD1 binders (e.g., an ISVDsuch as a Nanobody) of the invention are given in SEQ ID NOs: 9 to 40,57, 98, 99, 101, 102, 103, 104 or 105 and each of these amino acidsequences individually forms a further aspect of the invention.

As mentioned, in the invention, amino acid sequences in which position89 is T; or in which in which position 1 is E or D, position 11 is V,position 14 is P, position 52a is V, position 73 is P, S or Q, position74 is S, position 83 is R, position 89 is L and/or position 100a is F;or in which position 11 is V and position 89 is L (optionally insuitable combination with a 110K or 110Q mutation and/or a 112K or 112Qmutation, and in particular in combination with a 110K or 110Q mutation)are part of the present invention. In an embodiment of the invention,the amino acid sequence at position 11 is V and position 89 is L,optionally with a 110K or 110Q mutation.

Thus, in one preferred aspect, the invention relates to a PD1 binder(e.g., an immunoglobulin single variable domain (ISVD) such as aNanobody) having:

-   -   a CDR1 (according to Abm) that is the amino acid sequence        GSIASIHAMG (SEQ ID NO: 6); and    -   a CDR2 (according to Abm) that is the amino acid sequence        VITXSGGITY (SEQ ID NO: 7, wherein X is W or V); and    -   a CDR3 (according to Abm) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and also having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be        present as well as the CDRs, optionally comprising a W52aV        and/or W100aF mutation, are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (e.g.,        100%) (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        in which, in an embodiment of the invention:    -   the amino acid residue at position 1 is E or D;    -   the amino acid residue at position 11 is L or V;    -   the amino acid residue at position 14 is A or P;    -   the amino acid residue at position 52a is W or V;    -   the amino acid residue at position 73 is S, P, N or Q;    -   the amino acid residue at position 74 is A or S;    -   the amino acid residue at position 83 is K or R;    -   the amino acid residue at position 89 is T, L or I;    -   the amino acid residue at position 100a is W or F;    -   the amino acid residue at position 110 is T, K or Q (and is        preferably T); and    -   the amino acid residue at position 112 is S, K or Q (and in        preferably S).

In another preferred aspect, the invention relates to a PD1 binder(e.g., an immunoglobulin single variable domain (ISVD) such as aNanobody) having:

-   -   a CDR1 (according to Abm) that is the amino acid sequence        GSIASIHAMG (SEQ ID NO: 6); and    -   a CDR2 (according to Abm) that is the amino acid sequence        VITXSGGITY (SEQ ID NO: 7, wherein X is W or V); and    -   a CDR3 (according to Abm) that is the amino acid sequence        DKHQSSXYDY (SEQ ID NO: 5, wherein X is W or F);        and also having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 1 or 2 (in which any C-terminal extension that may be        present as well as the CDRs, optionally comprising a W52aV        and/or W100aF mutation, are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (e.g.,        100%) (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 52a,        73, 74, 83, 89, 100a, 110 and/or 112 required by the specific        aspect involved are not taken into account for determining the        degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 52a, 73, 74, 83, 89,        100a, 110 and/or 112 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 1 or 2 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I);        in which, for example, one or more of the following is true:    -   the amino acid residue at position 1 is E or D;    -   the amino acid residue at position 11 is V;    -   the amino acid residue at position 14 is P;    -   the amino acid residue at position 52a is V;    -   the amino acid residue at position 73 is S, P or Q;    -   the amino acid residue at position 74 is S;    -   the amino acid residue at position 83 is R;    -   the amino acid residue at position 89 is L;    -   the amino acid residue at position 100a is F;    -   the amino acid residue at position 110 is T, K or Q; or    -   the amino acid residue at position 112 is S, K or Q.

In one specific, but non-limiting aspect, the PD1 binders (e.g., an ISVDsuch as a Nanobody) of the invention comprise one of the following setsof mutations (i.e. mutations compared to the sequence of SEQ ID NO: 1 or2) at the positions mentioned (numbering according to Kabat):

-   -   11V in combination with 89L;    -   11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R,        89L, 100aF and 1E or 1D;    -   11V in combination with 14P, 74S, 83R, 89L and 1E or 1D;    -   11V in combination with 110K or 110Q;    -   11V in combination with 112K or 112Q;    -   11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R,        89L, 100aF, 110K or 110Q and 1E or 1D;    -   11V in combination with 14P, 52aV, 73S or 73Q or 73P, 74S, 83R,        89L, 100aF, 112K or 112Q and 1E or 1D;    -   11V in combination with 14P, 74S, 83R, 89L, 110K or 110Q and 1E        or 1D;    -   11V in combination with 14P, 74S, 83R, 89L, 112K or 112Q and 1E        or 1D;    -   11V in combination with 89L and 110K or 110Q; or    -   11V in combination with 89L and 112K or 112Q;        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 1, 2, 9-40, 57, 98, 99, 101, 102,        103, 104 or 105 (e.g., according to Abm) and have an overall        degree of sequence identity with the amino acid sequence of SEQ        ID NO: 1 or 2 that are as described herein.

In another specific, but non-limiting aspect, the PD1 binders (e.g., anISVD such as a Nanobody) of the invention comprise one of the followingsets of mutations (i.e. mutations compared to the sequence of SEQ ID NO:1 or 2) at the positions mentioned (numbering according to Kabat):

-   -   89L in combination with 11V;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF and 1E or 1D;    -   89L in combination with 11V, 14P, 74S, 83R and 1E or 1D;    -   89L in combination with 110K or 110Q;    -   89L in combination with 112K or 112Q;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF, 110K or 110Q and 1E or 1D;    -   89L in combination with 11V, 14P, 52aV, 73S or 73Q or 73P, 74S,        83R, 100aF, 112K or 112Q and 1E or 1D;    -   89L in combination with 11V, 14P, 74S, 83R, 110K or 110Q and 1E        or 1D;    -   89L in combination with 11V, 14P, 74S, 83R, 112K or 112Q and 1E        or 1D;    -   89L in combination with 11V and 110K or 110Q; or    -   89L in combination with 11V and 112K or 112Q;        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 1, 2, 9-40, 57, 98, 99, 101, 102,        103, 104 or 105 (e.g., according to Abm) and have an overall        degree of sequence identity with the amino acid sequence of SEQ        ID NO: 1 or 2 that are as described herein.

In another specific, but non-limiting aspect, the PD1 binders (e.g., anISVD such as a Nanobody) of the invention comprise one of the followingsets of mutations (i.e. mutations compared to the sequence of SEQ ID NO:1 or 2) at the positions mentioned (numbering according to Kabat):

-   -   110K or 110Q in combination with 11V;    -   110K or 110Q in combination with 89L;    -   110K or 110Q in combination with 11V and 89L;    -   110K or 110Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L and 100aF, and 1E or 1D;    -   110K or 110Q in combination with 11V, 14P, 74S, 83R, 89L and 1D;        or    -   110K or 110Q in combination with 11V, 14P, 74S, 83R, 89L, and        1E,        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 1, 2, 9-40, 57, 98, 99, 101, 102,        103, 104 or 105 (e.g., according to Abm) and have an overall        degree of sequence identity with the amino acid sequence of SEQ        ID NO: 1 or 2 that are as described herein.

In another specific, but non-limiting aspect, the PD1 binders (e.g., anISVD such as a Nanobody) of the invention comprise one or more of thefollowing sets of mutations (i.e. mutations compared to the sequence ofSEQ ID NO: 1 or 2) at the positions mentioned (numbering according toKabat):

-   -   112K or 112Q in combination with 11V;    -   112K or 112Q in combination with 89L;    -   112K or 112Q in combination with 11V and 89L;    -   112K or 112Q in combination with 11V, 14P, 52aV, 73S or 73Q or        73P, 74S, 83R, 89L and 100aF, and 1E or 1D; or    -   112K or 112Q in combination with 11V, 14P, 74S, 83R, 89L, and 1E        or 1D;        and have CDRs that are the same as the CDRs that are present in        the sequence of SEQ ID NO: 1, 2, 9-40, 57, 98, 99, 101, 102,        103, 104 or 105 (e.g., according to Abm) and have an overall        degree of sequence identity with the amino acid sequence of SEQ        ID NO: 1 or 2 that are as described herein.

In another aspect, the PD1 binders (e.g., an ISVD such as a Nanobody) ofthe invention comprise a T or L at position 89 and have CDRs that arethe same as the CDRs that are present in the sequence of SEQ ID NO: 1,2, 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 (e.g., according to Abm)and have an overall degree of sequence identity with the amino acidsequence of SEQ ID NO: 1 or 2 that are as described herein.

In another aspect, the PD1 binders (e.g., an ISVD such as a Nanobody) ofthe invention comprise a V at position 11 and an L at position 89 andhave CDRs that are the same as the CDRs that are present in the sequenceof SEQ ID NO: 1, 2, 9-40, 57, 98, 99, 101, 102, 103, 104 or 105 (e.g.,according to Abm) and have an overall degree of sequence identity withthe amino acid sequence of SEQ ID NO: 1 or 2 that are as describedherein.

As mentioned, the PD-1 binders of the invention according to the aboveaspects are preferably further such that they contain a suitablecombination of an E1D mutation, an L11V mutation, an A14P mutation, aW52aV mutation, an N73S or N73Q or N73P mutation, an A74S mutation, aK83R mutation, an I89L mutation and/or a W100aF mutation, and preferablya suitable combination of any two of these mutations, such as all ofthese mutations. When a PD-1 binder is monovalent or wherein the PD1binding moiety thereof is present at the N-terminal end of apolypeptide, it preferably also has an E1D mutation.

In another specific, but non-limiting aspect, the invention relates toan immunoglobulin single variable domain that comprises or consistsessentially of an amino acid sequence chosen from one of the followingamino acid sequences: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO: 57, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105.

In another specific, but non-limiting aspect, the invention relates toan immunoglobulin single variable domain that is or essentially consistsof an amino acid sequence chosen from one of the following amino acidsequences: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 39, SEQ ID NO: 40,SEQ ID NO: 57, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO: 104 and SEQ ID NO: 105.

Also, as already indicated herein, the amino acid residues of a PD1binder (e.g., an ISVD such as a Nanobody) are numbered according to thegeneral numbering for VHs given by Kabat et al. (“Sequence of proteinsof immunological interest”, US Public Health Services, NIH Bethesda,Md., Publication No. 91), as applied to VHH domains from Camelids in thearticle of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23;240 (1-2): 185-195; or referred to herein. It should be noted that, asis well known in the art for VH domains and for VHH domains, the totalnumber of amino acid residues in each of the CDRs may vary and may notcorrespond to the total number of amino acid residues indicated by theKabat numbering (that is, one or more positions according to the Kabatnumbering may not be occupied in the actual sequence, or the actualsequence may contain more amino acid residues than the number allowedfor by the Kabat numbering). This means that, generally, the numberingaccording to Kabat may or may not correspond to the actual numbering ofthe amino acid residues in the actual sequence. Generally, however,according to the numbering of Kabat and irrespective of the number ofamino acid residues in the CDRs, position 1 according to the Kabatnumbering corresponds to the start of FR1 and vice versa, position 36according to the Kabat numbering corresponds to the start of FR2 andvice versa, position 66 according to the Kabat numbering corresponds tothe start of FR3 and vice versa, and position 103 according to the Kabatnumbering corresponds to the start of FR4 and vice versa.].

Alternative methods for numbering the amino acid residues of VH domains,which methods can also be applied in an analogous manner to VHH domainsfrom Camelids and to Nanobodies, are the method described by Chothia etal. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and theso-called “contact definition”. However, in the present description,aspects and figures, the numbering according to Kabat as applied to VHHdomains by Riechmann and Muyldermans will be followed, unless indicatedotherwise.

These and other aspects, embodiments, advantages, applications and usesof the invention will become clear from the further description herein.

Accordingly, in a further aspect, the invention relates to polypeptidesor other chemical entities that comprise or essentially consist of atleast one (such as one, two or three) PD1 binding moieties describedherein.

PD1 binders (e.g., an ISVD such as a Nanobody) of the invention can befused to one or more other amino acid sequences, chemical entities ormoieties. These other amino acid sequences, chemical entities ormoieties can confer one or more desired properties to the resulting PD1binders of the invention and/or can alter the properties of theresulting PD1 binders of the invention in a desired manner, for exampleto provide the resulting PD1 binders of the invention with a desiredbiological and/or therapeutic activity (for example, to provide theresulting PD1 binders of the invention with affinity and preferablypotency against another therapeutically relevant target such that theresulting polypeptide becomes “bispecific” with respect to PD1 and thatother therapeutically relevant target such as CTLA4, LAG3, BTLA orCD27), to provide a desired half-life and/or to otherwise modify orimprove pharmacokinetic and/or pharmacodynamic properties, to target thePD1 binder to specific cells, tissues or organs (including cancer cellsand cancer tissues), to provide a cytotoxic effect and/or to serve as adetectable tag or label. Some non-limiting examples of such other aminoacid sequences, chemical entities or moieties are:

-   -   one or more suitable linkers (such as a 9GS, 15GS or 35GS linker        (any combination of 9, 15, 20 or 35 G and S amino acids such as,        for example, GGGGSGGGS (9GS linker; SEQ ID NO: 125),        GGGGSGGGGSGGGGSGGGGS (20GS linker; SEQ ID NO: 100) or        GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS linker; SEQ ID NO:        58)) or (GGGS)n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10);        and/or    -   one or more binding moieties, binding domains or binding units        that are directed against a therapeutically relevant target        other than PD1 (i.e. so as to provide a PD1 binder (e.g., an        ISVD such as a Nanobody) of the invention that is bispecific for        both PD1 and the other therapeutically relevant target, for        example, against a different epitope of PD1, CD27, LAG3, CTLA4,        BTLA, TIM3, ICOS, B7-H3, B7-H4, CD137, GITR, PD-L1, PD-L2, ILT1,        ILT2 CEACAM1, CEACAMS, TIM3, TIGIT, VISTA, ILT3, ILT4, ILT5,        ILT6, ILT7, ILT8, CD40, OX40, CD137, KIR2DL1, KIR2DL2, KIR2DL3,        KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, NKG2A,        NKG2C, NKG2E, IL-10, IL-17, TSLP); and/or    -   one or more binding domains or binding units that provide for an        increase in half-life (for example, a binding domain or binding        unit that can bind against a serum protein such as serum        albumin, e.g., human serum albumin), e.g., ALB11002; and/or    -   one or more binding domains or binding units that target the PD1        binder (e.g., an ISVD such as a Nanobody) to a desired cell,        tissue or organ (such as a cancer cell); and/or    -   one or more binding domains or binding units that provide for        increased specificity against PD1 (usually, these will be able        to bind to PD1 but will generally by themselves essentially not        be functional against PD1); and/or    -   a binding domain, binding unit or other chemical entity that        allows for the PD1 binder (e.g., an ISVD such as a Nanobody) to        be internalized into a desired cell (for example, an        internalizing anti-EGFR Nanobody as described in WO 05/044858);        and/or    -   a moiety that improves half-life such as a suitable        polyethyleneglycol group (i.e. PEGylation) or an amino acid        sequence that provides for increased half-life such as human        serum albumin or a suitable fragment thereof (i.e. albumin        fusion) or for example a serum albumin binding peptide as        described in WO 2008/068280; and/or    -   a payload such as a cytotoxic payload; and/or    -   a detectable label or tag, such as a radiolabel or fluorescent        label; and/or    -   a tag that can help with immobilization, detection and/or        purification of the PD1 binder (e.g., an ISVD such as a        Nanobody), such as a HIS (e.g., HHHHHH; SEQ ID NO: 93 or        HHHHHHHHHHHHHHHHHH; SEQ ID NO: 94) or FLAG tag (DYKDDDK (SEQ ID        NO: 95)) (e.g., FLAG₃); and/or    -   a tag that can be functionalized, such as a C-terminal GGC or        GGGC tag; and/or    -   a C-terminal extension X(n) (e.g., -Ala), which may be as        further described herein for the PD1 binders (e.g., an ISVD such        as a Nanobody) of the invention and/or as described in WO        12/175741 or PCT/EP2015/060643 (WO2015/173325).

The scope of the invention includes PD1 binder (e.g., an ISVD such as aNanobody) that include one or more parts or fragments of a (preferablyhuman) antibody (such as an Fc part or a functional fragment thereof orone or more constant domains) and/or from a Camelid heavy-chain onlyantibody (such as one or more constant domains).

LAG3 Binders

The present invention provides improved LAG3 binders, for example,improved anti-LAG3 ISVDs and more in particular improved LAG3Nanobodies, e.g., 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R,V89L); F0237611B09 (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L);F0237611B09(E1D,L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-ALB11002-A; orF0237611B09(E1D,L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-F0237611B09(L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-35GS-ALB11002-A.

As discussed the “LAG3 binders” of the present invention are any of themolecules described herein that bind to LAG3 (e.g., an ISVD such as aNanobody) as well as any multivalent or multispecific binder (e.g.,PD1/LAG3 binder) which includes such a molecule that is fused to anotherbinder. For example, a LAG3 binder may include a LAG3 binding moietyfused to a moiety that binds to PD1, CD27, CTLA4, HSA, BTLA, TIM3, ICOS,B7-H3, B7-H4, CD137, GITR, PD-L1, PD-L2, ILT1, ILT2 CEACAM1, CEACAM5,TIM3, TIGIT, VISTA, ILT3, ILT4, ILT5, ILT6, ILT7, ILT8, CD40, OX40,CD137, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1,KIR3DL2, KIR3DL3, NKG2A, NKG2C, NKG2E, IL-10, IL-17, TSLP. An individualLAG3 binder may be referred to as a LAG3 binding moiety if it is part ofa larger molecule, e.g., a multivalent molecule, wherein the LAG3binding moiety is fused to another binding moiety, such as F023700924 orF023700931.

LAG3 binders of the present invention include polypeptides which arevariants of polypeptides comprising the amino acid sequence of SEQ IDNO: 63 but which is mutated at position 1, 11, 14, 41 43, 62, 74, 83and/or 89.

As further described herein, the LAG3 binders of the inventionpreferably have the same combination of CDRs (i.e. CDR1, CDR2 and CDR3)as are present in 11B09 or in a binder comprising the sequence of 11B09(SEQ ID NO: 63). See Table B-1.

The present invention also includes LAG3 binders which are variants of11B09 which comprise an amino acid sequence as set forth below in TableB-2 below. The scope of the present invention includes LAG3 binders thatinclude CDR1, CDR2 and CDR3 of said varaints set forth below in TableB-2.

In addition, the present invention includes PD1/LAG3 binders comprisinga LAG3 binding moiety that includes CDR1, CDR2 and CDR3 or the aminoacid sequence of 11B09 or of one of its varaints set forth below inTable B-2.

TABLE B-1 LAG3 Binder 11B09. Description Sequence 11B09 (may beEVQLVESGGGLVQAGGSLRLSCAASGRTFS DYVNG WERQARGNEREFVA AISESGGRTreferred to herein HYADAVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCATTLLWWTSEYAPIKANDYD as ″F0237611B09″ Y WGQGTLVTVSS or ″611B09″)SEQ ID NO: 63

TABLE B-2 Sequence Optimized 11B09 LAG3 Binders. Description Sequence11B09 (L11V, A14P, R41P, EVQLVE SGGGVVQPGG SLRLSCAASG RTFS DYVMG WN43K, A62S, A74S, K83R, FRQAPGKERE FVA AISESGG RTHYADSVKG RFTISRDNSKV89L) NTLYLQMNSL RPEDTALYYC ATT LLWWTSE YAPIKANDYD Name: F023700842 YWGQGTLVTV SS SEQ ID NO: 64 CDR1 (SEQ ID NO: 65) GRTFS DYVMG  or  DYVMG(SEQ ID NO: 154; amino acids 6-10 of  SEQ ID NO: 65)CDR2 (SEQ ID NO: 66) AISESGGRTHYADXVKG; wherein X is A or S(e.g., AISESGGRTHYADAVKG (SEQ ID NO: 140) orAISESGGRTHYADSVKG (SEQ ID NO: 141))or AISESGGRTH (SEQ ID NO: 190, amino acids 1-10 of SEQ ID NO: 66)CDR3 (SEQ ID NO: 67) TLLWWTSEYAPIKANDYDY Monovalent SO (sequenceDVQLVESGGGVVQPGGSLRLSCAASGRTFSDYVMGWFRQAPGKEREFVA optimized) 611B09 +ALB11002 AISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCATName: F023701128 TLLWWTSEYAPIKANDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGDescription: SGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGF0237611B09 (E1D, L11V, A14P,MSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQR41P, N43K, A62S, A74S, K83R, MNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAV89L)-35GS-ALB11002-A Target: hLAG-3 SEQ ID NO: 96 Bivalent SO (sequenceDVQLVESGGGVVQPGGSLRLSCAASGRTFSDYVMGWERQAPGKEREEVA optimized) 611B09 +ALB11002 AISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCATName: F023700962 TLLWWTSEYAPIKANDYDY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGDescription: F0237611B09SGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFSDYV(E1D, L11V, A14P, R41P, N43K, MGWERQAPGKEREEVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQ A62S, A74S, K83R, V89L)-35GS-MNSLRPEDTALYYCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVSSGGG F0237611B09GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNS(L11V, A14P, R41P, N43K, A62S,LRLSCAASGETFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGA74S, K83R, V89L)-35GS-RFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS ALB11002-A ATarget: hLAG-3 SEQ ID NO: 97 Name: F023700594EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYVMGWFRQARGNEREEVADescription: F0237611B09-AISESGGRTHYADAVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAT 35GS-F0237611B09-35GS-TLLWWTSEYAPIKANDY DYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG ALB11002SGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYV Target: hLAG-3MGWERQARGNEREEVAAISESGGRTHYADAVKGRFTISRDNAKNTLYLQ SEQ ID NO: 127MNSLKPEDTAVYYCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGETFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS *CDRs of LAG3 bindersunderscored and/or bold.

The present invention includes embodiments wherein the LAG3 binder ofthe invention (e.g., in a PD1/LAG3 binder) includes one, two or three ofthe CDRs of a LAG3 binder set forth above in Table B-1 or B-2 (e.g., SEQID NO: 63, 64, 95, 96 or 97) wherein each comprises 0, 1, 2, 3, 4, or 5amino acid substitutions, e.g., conservative substitutions, and/orcomprises 100, 99, 98, 97, 96 or 95% sequence identity relative to theCDR sequences set forth in Table B-1 or B-2 wherein the LAG3 binder ofthe invention having such CDRs retains the ability to bind to LAG3. Inan embodiment of the invention, the first amino acid of a LAG3 binder ofthe present invention is E. In an embodiment of the invention, the firstamino acid of a LAG3 binder of the present invention is E or D.

The present invention includes the LAG3 binder F023700656 (11B09 (E1D);SEQ ID NO: 63 (E1D)).

The Kabat residue numbers for certain residues of the LAG3 binders setforth in Table B-1 or B-2 are shown in the sequence below:

-   EVQLVE SGGGV₁₁VQP₁₄GG SLRLSCAASG RTFSDYVMGW FRQAP₄₁GK₄₃ERE    FVAAISESGG RTHYADS₆₂VKG RFTISRDNS₇₄K NTLYLQMNSL R₈₃PEDTAL₈₉YYC    ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS (SEQ ID NO: 64). Optionally,    residue 1 is a D.

The present invention includes any LAG3 binder comprising the amino acidsequence of SEQ ID NO: 63, 64 or 127 (or having the LAG3 binder moietyof SEQ ID NO: 96 or 97) (as well as LAG3 binders having an E1D or D1Emutation) or an amino acid sequence comprising 80% or more (e.g., 85%,90%, 95%, 96%, 97%, 98% or 99%) amino acid sequence identity (i.e.,comparing the full amino acid sequences) wherein the LAG3 binder retainsthe ability to bind to LAG3 and, optionally, includes an HSA binder.

The present invention includes LAG3 binders, such as LAG3 ISVDs (e.g., aNanobodies), having CDR1, CDR2 and CDR3 of a binder comprising the aminoacid sequence set forth in SEQ ID NO: 63, 64, 96, 97 or 127 (or avariant thereof as described herein), e.g., comprising the followingCDRs:

-   -   a CDR1 that comprises the amino acid sequence GRTFSDYVMG (SEQ ID        NO: 65); and    -   a CDR2 that comprises the amino acid sequence AISESGGRTH (SEQ ID        NO: 66); and    -   a CDR3 that comprises the amino acid sequence        TLLWWTSEYAPIKANDYDY (SEQ ID NO: 67);        and, optionally, having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 63, 64, 96, 97 or 127 (in which any C-terminal        extension that may be present as well as the CDRs are not taken        into account for determining the degree of sequence identity) of        at least 85%, preferably at least 90%, more preferably at least        95% (in which the CDRs, any C-terminal extension that may be        present, as well as the mutations at positions 1, 11, 14, 41 43,        62, 74, 76, 83, 89, 100 and/or 105 are not taken into account        for determining the degree of sequence identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (as defined        herein, and not taking into account any of the mutations set        forth herein at position(s) 1, 11, 14, 41 43, 62, 74, 76, 83,        89, 100 and/or 105 that may be present and not taking into        account any C-terminal extension that may be present) with the        amino acid sequence of SEQ ID NO: 63, 64, 96, 97 or 127 (in        which said amino acid differences, if present, may be present in        the frameworks and/or the CDRs but are preferably present only        in the frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension as discussed herein, e.g., (X)_(n), in        which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5        (and preferably 1 or 2, such as 1); and each X is an (preferably        naturally occurring) amino acid residue that is independently        chosen, and preferably independently chosen from the group        consisting of alanine (A), glycine (G), valine (V), leucine (L)        or isoleucine (I).

The present invention also includes LAG3 binders fused to one or morehalf-life extenders, such as an ISVD that binds human serum albumin,e.g., ALB11002.

Multispecific Binders

The present invention includes PD1 binders and LAG3 binders that may befused in a single multivalent, multispecific molecule that binds to PD1and LAG3 (a PD1/LAG3 binder) and, in an embodiment of the invention,such binders are linked to one or more half-life extenders thatincreases the half-life of the binders in the body of a subject. In anembodiment of the invention, the half-life extender is an ISVD (e.g, aNanobody) that specifically binds to human serum albumin (HSA), e.g.,ALB11002. In an embodiment of the invention, the multispecific binder isF023700899; F023700931; F023701016; F023701017; F023700924; F023700969;F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8;F023701161; F023701166; F023701171; F023701176; F023701162; F023701167;F023701172; or F023701177 as described herein.

In an embodiment of the invention, the PD1/LAG3 binder comprises

-   (1) a PD1 binder comprising CDR1, CDR2 and CDR3 of any of the PD1    binders set forth in Table A-1 or A-2, optionally, each    independently comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid    substitutions (e.g., conservative mutations) or:-   a CDR1 comprising the amino acid sequence IHAMG (SEQ ID NO: 3) or    GSIASIHAMG (SEQ ID NO: 6);-   a CDR2 comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID    NO: 4; wherein X is W or V, e.g., W) or VITXSGGITY (SEQ ID NO: 7;    wherein Xis W or V, e.g., W); and-   a CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5,    wherein X is W or F, e.g., W);-   and-   (2) a LAG3 binder comprising CDR1, CDR2 and CDR3 of any of the LAG3    binders set forth in Table B-1 or B-2, optionally, each    independently comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid    substitutions (e.g., conservative mutations) or:-   a CDR1 comprising the amino acid sequence GRTFSDYVMG (SEQ ID NO:    65);-   a CDR2 comprising the amino acid sequence AISESGGRTH (SEQ ID NO:    66); and-   a CDR3 comprising the amino acid sequence TLLWWTSEYAPIKANDYDY (SEQ    ID NO: 67),-   and, optionally, a half-life extender and/or a C-terminal extender,    for example, an HSA binder as set forth herein and/or, optionally a    C-terminal extender such as Alanine.

In an embodiment of the invention, the PD1 binder is as set forth aboveunder “PD1 Binders”, e.g., comprising the amino acid sequence of SEQ IDNO: 57, 98, 99, 103, 104 or 105 (optionally wherein residue 1 is E orD). In an embodiment of the invention, the LAG3 binder is as set forthabove under “LAG3 Binders”, e.g., comprising the amino acid sequence ofSEQ ID NO: 63, 64 or 95 (optionally wherein residue 1 is E or D).

Multispecific binders may include a PD1 and LAG3 and, optionally, an HSAbinder as well as one or more binders that bind to an additional antigensuch as, CD27, CTLA4, BTLA, TIM3, ICOS, B7-H3, B7-H4, CD137, GITR,PD-L1, PD-L2, ILT1, ILT2 CEACAM1, CEACAM5, TIM3, TIGIT, VISTA, ILT3,ILT4, ILT5, ILT6, ILT7, ILT8, CD40, OX40, CD137, KIR2DL1, KIR2DL2,KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, NKG2A,NKG2C, NKG2E, IL-10, IL-17 or TSLP.

When the PD1 and/or LAG3 binders (e.g., an ISVD such as a Nanobody)contain one or more further binding domains or binding units (e.g. afurther essentially non-functional binding domain or binding unitagainst PD1 and/or LAG3 that provides for increased specificity againstPD1 and/or LAG3, a binding domain or binding unit against a therapeutictarget other than PD1 and/or LAG3 (e.g., CTLA4, BTLA or CD27), a bindingdomain or binding unit against a target such as human serum albumin thatprovides for increased half-life, and/or a binding domain or bindingunit that targets the PD1 and/or LAG3 binder to a specific cell, tissueor organ and/or that allows for the PD1 and/or LAG3 binder to beinternalized into a cell), these other binding domains or binding unitspreferably comprise one or more ISVDs (e.g., Nanobodies), and morepreferably are all ISVDs. For example and without limitation, these oneor more further binding domains or binding units can be one or moreNanobodies (including a VHH, a humanized VHH and/or a camelized VHs suchas camelized human VHs), a (single domain) antibody is a VH domain orthat is derived from a VH domain, a dAb that is or essentially consistsof a VH domain or that is derived from a VH domain, or even a (single)domain antibody or a dAb that is or essentially consists of VL domain.In particular, these one or more binding domains or binding units, whenpresent, may comprise one or more Nanobodies, and more in particular areall Nanobodies. In an embodiment of the invention, the ISVD (e.g.,Nanobody) that binds a target other than PD1 and/or LAG3 binds toanother target such as HSA, CTLA-4, BTLA or CD27.

When a PD1 and/or LAG3 binder (e.g., an ISVD such as a Nanobody) of theinvention has an ISVD (e.g., Nanobody) at its C-terminal end (e.g.,which C-terminal ISVD binds to PD1 and/or LAG3 or may for example be, ifpresent in the polypeptide, a further essentially non-functional ISVDagainst PD1 and/or LAG3 that provides for increased specificity againstPD1 and/or LAG3, an ISVD against a therapeutic target other than PD1and/or LAG3, an ISVD against a target such as human serum albumin thatprovides for increased half-life, or an ISVD that targets the PD1 and/orLAG3 binder to a specific cell, tissue or organ and/or that allows forthe PD1 and/or LAG3 binder to be internalized into a cell), then the PD1and/or LAG3 binder (i.e. comprising said C-terminal ISVD) preferably hasa C-terminal extension X(n) (e.g., -Ala), which C-terminal extension maybe as described herein for the PD1 and/or LAG3 binders of the inventionand/or as described in WO 12/175741 or PCT/EP2015/060643(WO2015/173325).

When a PD1 and/or LAG3 binder contains, in addition to the one or moremoieties that bind to PD1 and/or LAG3, any further ISVDs (e.g.,Nanobodies) (which one or more further ISVDs may, as mentioned, be afurther essentially non-functional ISVD against PD1 and/or LAG3 thatprovides for increased specificity against PD1 and/or LAG3, an ISVDagainst a therapeutic target other than PD1 and/or LAG3, an ISVD againsta target such as human serum albumin that provides for increasedhalf-life, and/or an ISVD that targets the polypeptide of the inventionto a specific cell, tissue or organ and/or that allows for thepolypeptide of the invention to be internalized into a cell), and wheresuch further ISVDs are Nanobodies or are ISVDs that are, or thatessentially consist of and/or that are derived from VH sequences, thenaccording to a preferred aspect of the invention said one or more (andpreferably all) of such ISVDs present in the PD1 and/or LAG3 binder willcontain within their sequence one or more framework mutations thatreduce binding by pre-existing antibodies. In particular, according tothis aspect of the invention, such further ISVDs may contain a suitablecombination of amino acid residues/mutations at positions 1, 11, 14, 74,83, 89, 110 and/or 112 that are as described in PCT/EP2015/060643(WO2015/173325) and/or that essentially are as described herein for thePD1 and/or LAG3 binders of the invention. In one specific aspect, whenthe PD1 and/or LAG3 binder has such an ISVD at its C-terminal end, thensaid ISVD that is present at and/or forms the C-terminus has suchframework mutations that reduce binding by pre-existing antibodies; andsaid C-terminal ISVD will preferably also have a C-terminal extensionX(n) (e.g., -Ala) as described herein.

When a PD1 and/or LAG3 binder is to have an increased half-life (i.e.compared to a monovalent binder of the invention that lacks a half-lifeextender such as ALB11002), the PD1 and/or LAG3 binder preferablycontains at least one (e.g., one) ISVD (e.g., a Nanobody) that providesfor such increased half-life (e.g., ALB11002). Such an ISVD is, in anembodiment of the invention, directed against a suitable serum proteinsuch as transferrin or against (human) serum albumin. In particular,such an ISVD or Nanobody may be a (single) domain antibody or dAbagainst human serum albumin as described in for example EP 2 139 918, WO2011/006915, WO 2012/175400, WO 2014/111550 and may in particular be aserum albumin binding Nanobody as described in WO 2004/041865, WO2006/122787, WO 2012/175400 or PCT/EP2015/060643 (WO2015/173325).Particularly preferred serum albumin binding ISVDs are the NanobodyAlb-1 (see WO 2006/122787) or its humanized variants such as Alb-8 (WO2006/122787, SEQ ID NO: 62), Alb-23 (WO 2012/175400, SEQ ID NO: 1) andother humanized (and preferably also sequence-optimized) variants ofAlb-1 and/or variants of Alb-8 or Alb-23 (or more generally ISVDs thathave essentially the same CDRs as Alb-1, Alb-8 and Alb-23).

As discussed the “HSA binders” of the present invention any of themolecules, such as those described herein, that bind to HSA (e.g., anISVD such as a Nanobody) as well as any multivalent or multispecificbinder which includes such a molecule that is fused to another binder.An individual HSA binder may be referred to as a HSA binding moiety ifit is part of a larger molecule, e.g., a multivalent molecule, whereinthe HSA binding moiety is fused to another binding moiety.

In an embodiment of the invention, the half-life extender is an ISVD(e.g., Nanobody) that binds to human serum albumin, e.g., ALB11002 assummarized below in Table C.

As further described herein, the HSA binders of the invention preferablyhave the same combination of CDRs (i.e. CDR1, CDR2 and CDR3) as arepresent in ALB11002 or comnprising the sequence of ALB11002 (SEQ ID NO:59). See Table C.

The present invention also includes HSA binders which are variants ofALB11002 which comprise an amino acid sequence as set forth below inTable C below. The scope of the present invention includes HSA bindersthat include CDR1, CDR2 and CDR3 of said varaints set forth below inTable C.

In addition, the present invention includes PD1/LAG3 binders comprisingan HSA binding moiety that includes CDR1, CDR2 and CDR3 or the aminoacid sequence of ALB11002 or of one of its varaints set forth below inTable C.

TABLE C Human Serum Albumin (HSA) Nanobody ALB11002 SEQ ID NODescription Sequence 59 ALB11002 (may be referredEVQLVESGGG XVQPGNSLRL SCAASGFTFS to herein as ″ALB201″) SFGMSWVRQA PGKGLEWVS S ISGSGSDTLY ADSVKGRFTI SRDNAKTTLY LQMNSLRPED TAXYYCTIGG SLSR SSQGTL VTVSSA; wherein X at residues 11 and 93 are L or V 60CDR1 GFTFSSFGMS or SFGMS (SEQ ID NO: 151;amino acids 6-10 of SEQ ID NO: 60) 61 CDR2SISGSGSDTLYADSVKG or SISGSGSDTL (SEQ ID NO:152; amino acids 1-10 of SEQ ID NO: 61) 62 CDR3 GGSLSR *Optionally,ALB11002 lacks the C-terminal Alanine. Optionally, the HSA bindercomprises the amino acid sequence set forth in SEQ ID NO: 59 but whichcomprises an E1D, V11L and/or an L93V mutation, e.g., comprising theamino acid sequence:EVQLVESGGGVVQPGNSLRLSCAASGETFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS(SEQ ID NO: 59)

In an embodiment of the invention, the first amino acid of a HSA binderof the present invention is E. In an embodiment of the invention, thefirst amino acid of a HSA binder of the present invention is D.

The present invention includes embodiments wherein one, two or three ofthe CDRs of a HSA binder each comprises 0, 1, 2, 3, 4, or 5 amino acidsubstitutions, e.g., conservative substitutions, and/or comprises 99,98, 97, 96 or 95% sequence identity relative to the CDR sequences setforth in Table C, wherein the HSA binder having such CDRs retain theability to bind to HSA.

In an embodiment of the invention, the half-life extender is an HSA ISVD(e.g., a Nanobody) comprising:

-   -   a CDR1 that comprises the amino acid sequence GFTFSSFGMS (SEQ ID        NO: 60); and    -   a CDR2 that comprises the amino acid sequence SISGSGSDTL (SEQ ID        NO: 61); and    -   a CDR3 that comprises the amino acid sequence GGSLSR (SEQ ID NO:        62);        and, optionally, having:    -   a degree of sequence identity with the amino acid sequence of        SEQ ID NO: 59 (in which any C-terminal extension that may be        present as well as the CDRs are not taken into account for        determining the degree of sequence identity) of at least 85%,        preferably at least 90%, more preferably at least 95% (in which        the CDRs, any C-terminal extension that may be present are not        taken into account for determining the degree of sequence        identity);        and/or    -   no more than 7, such as no more than 5, preferably no more than        3, such as only 3, 2 or 1 “amino acid differences” (not taking        into account any C-terminal extension that may be present) with        the amino acid sequence of SEQ ID NO: 59 (in which said amino        acid differences, if present, may be present in the frameworks        and/or the CDRs but are preferably present only in the        frameworks and not in the CDRs);        and optionally having:    -   a C-terminal extension (X)_(n), in which n is 1 to 10,        preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or        2, such as 1); and each X is an (preferably naturally occurring)        amino acid residue that is independently chosen, and preferably        independently chosen from the group consisting of alanine (A),        glycine (G), valine (V), leucine (L) or isoleucine (I).

Such a human serum albumin binding ISVD (e.g., Nanobody), when present,may contain within its sequence one or more framework mutations thatreduce binding by pre-existing antibodies. In particular, when such aserum albumin binding ISVD is a Nanobody or a (single) domain antibodythat is, essentially consist of and/or is derived from a VH domain, theserum albumin binding ISVD may contain (a suitable combination of) aminoacid residues/mutations at positions 11, 89, 110 and/or 112 that are asdescribed in PCT/EP2015/060643 (WO2015/173325) and/or that essentiallyare as described herein for the PD1 binders of the invention. Forexample, PCT/EP2015/060643 (WO2015/173325) describes a number ofvariants of Alb-1, Alb-8 and Alb-23 that contain amino acidresidues/mutations at positions 11, 89, 110 and/or 112 that reducebinding by pre-existing antibodies that can be used in the polypeptidesof the invention.

When such a serum albumin binding ISVD (e.g., Nanobody) is present atthe C-terminal end of a PD1 and/or LAG3 binder, the serum albuminbinding ISVD (and as a result, the PD1 and/or LAG3 binder of theinvention) preferably has a C-terminal extension X(n), which C-terminalextension may be as described herein for the PD1 and/or LAG3 binders ofthe invention and/or as described in WO 12/175741 or PCT/EP2015/060643(WO2015/173325). For example, the C-terminal extension may be a singleAlanine residue. It also preferably has mutations that reduce thebinding of pre-existing antibodies, like (a suitable combination of) theamino acid residues/mutations at positions 11, 89, 110 and/or 112described in PCT/EP2015/060643 (WO2015/173325).

However, as mentioned, other means of increasing the half-life of apolypeptide of the invention (such as PEGylation, fusion to humanalbumin or a suitable fragment thereof, or the use of a suitable serumalbumin-binding peptide), are also included in the scope of theinvention.

Generally, when a PD1 and/or LAG3 binder of the invention has increasedhalf-life (e.g. through the presence of a half-life increasing ISVD(e.g., Nanobody) or any other suitable way of increasing half-life), theresulting PD1 and/or LAG3 binder of the invention preferably has ahalf-life (as defined herein) that is at least 2 times, preferably atleast 5 times, for example at least 10 times or more than 20 times,greater than the half-life of a PD1 and/or LAG3 binder of the inventionlacking the a half-life extender (as measured in either in man and/or asuitable animal model, such as mouse or cynomolgus monkey). Inparticular, a PD1 and/or LAG3 binder of the invention preferably has ahalf-life (as defined herein) in human subjects of at least 1 day,preferably at least 3 days, more preferably at least 7 days, such as atleast 10 days.

It will be clear from the disclosure herein that PD1 binders and LAG3binders that are based on one or more ISVDs (e.g., Nanobodies) can havedifferent “formats”, i.e. essentially be monovalent, bivalent ortrivalent, can be monospecific, bispecific, trispecific etc., and can bebiparatopic (as defined herein and in for example WO2009/68625). Forexample, a PD1 binder or LAG3 binder of the invention can be:

-   -   monovalent, i.e. comprising a single PD1 or LAG3 binding moiety        (e.g., an ISVD such as a Nanobody). As mentioned, when used in        monovalent format, a PD1 binder or LAG3 binder of the invention        preferably has a C-terminal extension X(n) as further described        herein. Such a PD1 binder or LAG3 binder of the invention may        also be half-life extended;    -   can be bivalent or trivalent and monospecific. For example, in        an embodiment of the invention, such a PD1 binder or LAG3 binder        of the invention comprises two or more ISVDs (e.g., Nanobodies)        against PD 1 or against LAG3, respectively, which may be the        same or different; and when different, may be directed against        the same epitope of PD 1 or LAG3, or against different epitopes        on PD 1 or LAG3 (in the latter case, so as to provide a        biparatopic or multiparatopic PD 1 binder or LAG3 binder of the        invention). Such a PD 1 binder or LAG3 binder of the invention        may also be half-life extended;    -   can be bivalent, trivalent (or multivalent) and bispecific or        trispecific (or multispecific). For example, in an embodiment of        the invention, such a PD1 binder of the invention will be        directed against PD 1 and at least one other target such as, for        example, CTLA-4, LAG-3, BTLA and/or CD27; e.g., comprising a PD1        binding moiety and a CTLA4 binding moiety; a PD 1 binding moiety        and a BTLA binding moiety; a PD 1 binding moiety and a LAG3        binding moiety (i.e., a PD1/LAG3 binder); or a PD1 binding        moiety, a LAG3 binding moiety and a BTLA binding moiety. As        described herein, said other target may for example be another        therapeutically relevant target (i.e. other than PD1) so as to        provide a PD1 binder of the invention that is bispecific with        regards to PD1 and said other therapeutic target. Said other        target may also be a target that provides for increased        half-life (such as human serum albumin), so as to provide a PD1        binder of the invention that has increased half-life. As also        mentioned herein, such other target may allow also for the PD1        binder of the invention to be targeted to specific cells,        tissues or organs or may allow for the PD1 binder of the        invention to be internalized into a cell. It is also possible to        combine these approaches/ISVDs, for example to provide a PD1        binder of the invention that is bispecific for PD1 and for at        least one other therapeutically relevant target and that is        half-life extended.

Again, preferably, when these PD1 binders and LAG3 binders include oneor more ISVDs (e.g., Nanobodies) other than a PD1 binding moiety or LAG3binding moiety, at least one and preferably all of these other ISVDswill contain within its sequence one or more framework mutations thatreduce binding by pre-existing antibodies (such as, in particular, acombination of amino acid residues/mutations at positions 1, 11, 14,52a, 73, 74, 83, 89, 100a, 110 and/or 112 that is as described hereinfor the PD1 binders and LAG3 binders of the invention and/or asgenerally described in PCT/EP2015/060643 (WO2015/173325)). Also, whensuch PD1 binders or LAG3 binders of the invention have a PD1 bindingmoiety or LAG3 binding moiety, respectively, at their C-terminal end,then said C-terminal PD1 binding moiety or LAG3 binding moiety (and as aresult, the PD1 binder or LAG3 binder of the invention) will preferablyhave a C-terminal extension X(n) as described herein. Similarly, whensuch PD1 binders LAG3 binders of the invention have another ISVD attheir C-terminal end (i.e. not a PD1 binding moiety or LAG3 bindingmoiety, but for example a half-life extending ISVD), then saidC-terminal ISVD (and, as a result, the PD1 binder or LAG3 binder of theinvention will preferably has a C-terminal extension X(n) as describedherein and/or will contain within its sequence one or more frameworkmutations that reduce binding by pre-existing antibodies (again, asfurther described herein and in PCT/EP2015/060643 (WO2015/173325)).

As will be clear to the skilled person, when a PD1 binder or LAG3 binder(e.g., an ISVD such as a Nanobody) of the invention is intended fortopical use (i.e. on the skin or in the eye) or is for example meant tohave a (localized) therapeutic action somewhere in for example the GItract (i.e. after oral administration or administration by suppository)or in the lungs (i.e. after administration by inhalation) or isotherwise meant to be directly applied to its intended place of action(for example, by direct injection), a PD1 binder or LAG3 binder of theinvention will usually not require half-life extension. Also, fortreatment of certain acute conditions or indications, it may bepreferable not to have a prolonged half-life. In these cases, the use ofa monovalent PD1 binder or LAG3 binder of the invention or of anotherPD1 binder or LAG3 binder of the invention (comprising a PD1 binderor orLAG3 binder) without half-life extension, for example, a PD1 binder orLAG3 binder of the invention that is bivalent or biparatopic withrespect to PD1 or LAG3, is preferred.

Some preferred, but non-limiting examples of such PD1 binders and orLAG3 binder of the invention are schematically represented in Table D-1aand D-1b below, and each of these forms a further aspect of theinvention. Other examples of suitable polypeptides of the inventionwithout half-life extension will be clear to the skilled person based onthe disclosure herein.

TABLE D-1a Schematic Representation of Some PD1 and/or LAG3 Binders ofthe Invention Without a Half-Life Extending ISVD. [PD-1 binder of theinvention] [PD-1 binder of the invention]-X(n) [PD-1 binder of theinvention]-[PD-1 binder of the invention]-[Other]- [Other] [PD-1 binderof the invention]-[PD-1 binder of the invention]-[Other]- [Other] [PD-1binder of the invention]-[PD-1 binder of the invention]-[Other]-[Other]-X(n) [PD-1 binder of the invention]-[PD-1 binder of theinvention]-[Other]- [Other]-X(n) [PD-1 binder of the invention]-[PD-1binder of the invention]-X(n) [PD-1 binder of the invention]-[Other][PD-1 binder of the invention]-[Other]-X(n) [Other]-[PD-1 binder of theinvention] [Other]-[PD-1 binder of the invention]-X(n) [PD-1 binder ofthe invention]-[Targeting unit] [Targeting unit]-[PD-1 binder of theinvention] [PD-1 binder of the invention]-[Targeting unit]-X(n)[Targeting unit]-[PD-1 binder of the invention]-X(n) [PD-1 binder of theinvention]-[PD-1 binder of the invention]- [Targeting unit] [PD-1 binderof the invention]-[PD-1 binder of the invention]- [Targeting unit]-X(n)[Targeting unit]-[PD-1 binder of the invention]-[PD-1 binder of theinvention] [Targeting unit]-[PD-1 binder of the invention]-[PD-1 binderof the invention]-X(n) [PD-1 binder of the invention] [PD-1 binder ofthe invention]-X(n) [PD-1 binder of the invention]-[PD-1 binder of theinvention] [PD-1 binder of the invention]-[PD-1 binder of theinvention]-X(n) [PD-1 binder of the invention]-[Other] [PD-1 binder ofthe invention]-[Other]-X(n) [Other]-[PD-1 binder of the invention][Other]-[PD-1 binder of the invention]-X(n) [PD-1 binder of theinvention]-[Targeting unit] [Targeting unit]-[PD-1 binder of theinvention] [PD-1 binder of the invention]-[Targeting unit]-X(n)[Targeting unit]-[PD-1 binder of the invention]-X(n) Legend: “[PD1binder of the invention]” represents a PD1 binding domain or moiety orunit such as the ISVD 102C12 (E1D, L11V, A14P, A74S, K83R, I89L) asdescribed herein “—” represents either a direct covalent linkage or asuitable linker, such as a 9GS, 15GS or 35GS linker “X(n)” represents aC-terminal extension as defined herein such as a single alanine residue.“[Other]” represents a binding domain or binding unit (e.g., an ISVDsuch as a Nanobody) against an epitope that is different from the PD1binder, e.g., one or more CTLA4, BTLA, LAG3 and/or CD27 ISVDs such asthe LAG3 ISVD 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L) asdescribed herein “[Targeting unit]” represents a binding domain orbinding unit (and in particular ISVD such as a Nanobody) that targetsthe polypeptide of the invention to a specific cell, tissue or organ

TABLE D-1b Schematic Representation of Some PD1 and/or LAG3 Binders ofthe Invention Without a Half-Life Extending ISVD. [LAG3 binder of theinvention] [LAG3 binder of the invention]-X(n) [LAG3 binder of theinvention]-[LAG3 binder of the invention]-[Other]- [Other] [LAG3 binderof the invention]-[LAG3 binder of the invention]-[Other]- [Other] [LAG3binder of the invention]-[LAG3 binder of the invention]-[Other]-[Other]-X(n) [LAG3 binder of the invention]-[LAG3 binder of theinvention]-[Other]- [Other]-X(n) [LAG3 binder of the invention]-[LAG3binder of the invention]-X(n) [LAG3 binder of the invention]-[Other][LAG3 binder of the invention]-[Other]-X(n) [Other]-[LAG3 binder of theinvention] [Other]-[LAG3 binder of the invention]-X(n) [LAG3 binder ofthe invention]-[Targeting unit] [Targeting unit]-[LAG3 binder of theinvention] [LAG3 binder of the invention]-[Targeting unit]-X(n)[Targeting unit]-[LAG3 binder of the invention]-X(n) [LAG3 binder of theinvention]-[LAG3 binder of the invention]- [Targeting unit] [LAG3 binderof the invention]-[LAG3 binder of the invention]- [Targeting unit]-X(n)[Targeting unit]-[LAG3 binder of the invention]-[LAG3 binder of theinvention] [Targeting unit]-[LAG3 binder of the invention]-[LAG3 binderof the invention]-X(n) [LAG3 binder of the invention] [LAG3 binder ofthe invention]-X(n) [LAG3 binder of the invention]-[LAG3 binder of theinvention] [LAG3 binder of the invention]-[LAG3 binder of theinvention]-X(n) [LAG3 binder of the invention]-[Other] [LAG3 binder ofthe invention]-[Other]-X(n) [Other]-[LAG3 binder of the invention][Other]-[LAG3 binder of the invention]-X(n) [LAG3 binder of theinvention]-[Targeting unit] [Targeting unit]-[LAG3 binder of theinvention] [LAG3 binder of the invention]-[Targeting unit]-X(n)[Targeting unit]-[LAG3 binder of the invention]-X(n) Legend: “[LAG3binder of the invention]” represents a LAG3 binding domain or moiety orunit such as the ISVD 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R,V89L) as described herein “—” represents either a direct covalentlinkage or a suitable linker, such as a 9GS, 15GS or 35GS linker “X(n)”represents a C-terminal extension as defined herein such as a singlealanine residue. “[Other]” represents a binding domain or binding unit(e.g., an ISVD such as a Nanobody) against an epitope that is differentfrom the PD1 binder, e.g., one or more CTLA4, BTLA, LAG3 and/or CD27ISVDs such as the PD1 ISVD 102C12 (E1D, L11V, A14P, A74S, K83R, I89L) asdescribed herein “[Targeting unit]” represents a binding domain orbinding unit (and in particular ISVD such as a Nanobody) that targetsthe polypeptide of the invention to a specific cell, tissue or organ

As will be clear to the skilled person, when a PD1 binder or LAG3 binder(e.g., comprising an ISVD such as a Nanobody) of the invention isintended for systemic administration and/or for prevention and/ortreatment of a chronic disease or disorder, it will usually be preferredthat said PD1 binder or LAG3 binder of the invention has increasedhalf-life (as defined herein). More preferably, such a PD1 binder orLAG3 binder of the invention will contain a half-life extending ISVD(e.g., Nanobody) such as, preferably, an ISVD and in particular aNanobody binding to human serum albumin (as described herein).

Some preferred, but non-limiting examples of such PD1 binder or LAG3binder of the invention are schematically represented in Table D-2a andD-2b below, and each of these forms a further aspect of the invention.Other examples of a suitable PD1 binder or LAG3 binder of the inventionwith half-life extension will be clear to the skilled person based onthe disclosure herein. Generally, for polypeptides of the invention withhalf-life extension, the presence of a C-terminal extension is muchpreferred.

TABLE D-2a Schematic Representation of Some PD1 and/or LAG3 Binders ofthe Invention with a Half-Life Extending ISVD. [PD-1 binder of theinvention]-[HLE] [HLE]-[PD-1 binder of the invention] [PD-1 binder ofthe invention]-[HLE]-X(n) [HLE]-[PD-1 binder of the invention]-X(n)[PD-1 binder of the invention]-[PD-1 binder of the invention]-[HLE][PD-1 binder of the invention]-[HLE]-[PD-1 binder of the invention][HLE]-[PD-1 binder of the invention]-[PD-1 binder of the invention][PD-1 binder of the invention]-[PD-1 binder of the invention]-[HLE]-X(n)[PD-1 binder of the invention]-[HLE]-[PD-1 binder of the invention]-X(n)[HLE]-[PD-1 binder of the invention]-[PD-1 binder of the invention]-X(n)[PD-1 binder of the invention]-[Other]-[HLE] [PD-1 binder of theinvention]-[PD-1 binder of the invention]-[Other]- [Other]-[HLE] [PD-1binder of the invention]-[Other]-[HLE]-X(n) [PD-1 binder of theinvention]-[PD-1 binder of the invention]-[Other]- [Other]-[HLE]-X(n)[PD-1 binder of the invention]-[HLE]-[Other] [HLE]-[PD-1 binder of theinvention]-[Other] [HLE]-[Other]-[PD-1 binder of the invention][Other]-[PD-1 binder of the invention]-[HLE] [Other]-[HLE]-[PD-1 binderof the invention] [PD-1 binder of the invention]-[Other]-[HLE]-X(n)[PD-1 binder of the invention]-[HLE]-[Other]-X(n) [HLE]-[PD-1 binder ofthe invention]-[Other]-X(n) [HLE]-[Other]-[PD-1 binder of theinvention]-X(n) [Other]-[PD-1 binder of the invention]-[HLE]-X(n)[Other]-[HLE]-[PD-1 binder of the invention]-X(n) [PD-1 binder of theinvention]-[Targeting unit]-[HLE] [PD-1 binder of theinvention]-[HLE]-[Targeting unit] [HLE]-[PD-1 binder of theinvention]-[Targeting unit] [Targeting unit]-[PD-1 binder of theinvention]-[HLE] [Targeting unit]-[HLE]-[PD-1 binder of the invention][HLE]-[Targeting unit]-[PD-1 binder of the invention] [PD-1 binder ofthe invention]-[Targeting unit]-[HLE]-X(n) [PD-1 binder of theinvention]-[HLE]-[Targeting unit]-X(n) [HLE]-[PD-1 binder of theinvention]-[Targeting unit]-X(n) [Targeting unit]-[PD-1 binder of theinvention]-[HLE]-X(n) [Targeting unit]-[HLE]-[PD-1 binder of theinvention]-X(n) [HLE]-[Targeting unit]-[PD-1 binder of theinvention]-X(n) [PD-1 binder of the invention]-[PD-1 binder of theinvention]- [Targeting unit]-[HLE] [PD-1 binder of the invention]-[PD-1binder of the invention]- [HLE]-[Targeting unit] [PD-1 binder of theinvention]-[HLE]-[PD-1 binder of the invention]-[Targeting unit][HLE]-[PD-1 binder of the invention]-[PD-1 binder of theinvention]-[Targeting unit] [PD-1 binder of the invention]-[PD-1binderof the invention]- [Targeting unit]-[HLE]-X(n) [PD-1 binder of theinvention]-[PD-1 binder of the invention]- [HLE]-[Targeting unit]-X(n)[PD-1 binder of the invention]-[HLE]-[PD-1 binder of the invention]-[Targeting unit]-X(n) [HLE]-[PD-1 binder of the invention]-[PD-1 binderof the invention]- [Targeting unit]-X(n) [Targeting unit]-[PD-1 binderof the invention]-[PD-1 binder of the invention]-[HLE] [Targetingunit]-[PD-1 binder of the invention]-[HLE]-[PD-1 binder of theinvention] [Targeting unit]-[HLE]-[PD-1 binder of the invention]-[PD-1binder of the invention] [HLE]-[Targeting unit]-[PD-1 binder of theinvention]-[PD-1 binder of the invention] [Targeting unit]-[PD-1 binderof the invention]-[PD-1 binder of the invention]-[HLE]-X(n) [Targetingunit]-[PD-1 binder of the invention]-[HLE]-[PD-1 binder of theinvention]-X(n) [Targeting unit]-[HLE]-[PD-1 binder of theinvention]-[PD-1 binder of the invention]-X(n) [HLE]-[Targetingunit]-[PD-1 binder of the invention]-[PD-1 binder of the invention]-X(n)[PD-1 binder of the invention]-[HLE] [HLE]-[PD-1 binder of theinvention] [PD-1 binder of the invention]-[HLE]-X(n) [HLE]-[PD-1 binderof the invention]-X(n) [PD-1 binder of the invention]-[PD-1 binder ofthe invention]-[HLE] [PD-1 binder of the invention]-[HLE]-[PD-1 binderof the invention] [HLE]-[PD-1 binder of the invention]-[PD-1 binder ofthe invention] [PD-1 binder of the invention]-[PD-1 binder of theinvention]-[HLE]-X(n) [PD-1 binder of the invention]-[HLE]-[PD-1 binderof the invention]-X(n) [HLE]-[PD-1 binder of the invention]-[PD-1 binderof the invention]-X(n) [PD-1 binder of the invention]-[Other]-[HLE]Legend: “[PD1 binder of the invention]” represents a PD1 binding domainor binding unit such as the PD1 ISVD 102C12 (E1D, L11V, A14P, A74S,K83R, I89L) as described herein “—” represents either a direct covalentlinkage or a suitable linker, such as a 9GS, 15GS or 35GS linker “X(n)”represents a C-terminal extension as defined herein such as a singlealanine residue. “[HLE]” represents a half-life extending binding domainor binding unit (and in particular a half-life extending ISVD, such as aNanobody), such as an ISVD (and in particular Nanobody) against (human)serum albumin such as the anti-HSA ISVD ALB11002 as described herein;“[Other]” represents a binding domain or binding unit (e.g., an ISVDsuch as a Nanobody) against an epitope that is different from the PD1binder, e.g., one or more CTLA4, BTLA, LAG3 and/or CD27 ISVDs, such asthe LAG3 the ISVD 11B09 (L11V, A14P, R41P, N43K, A62S, A74S, K83R, V89L)as described herein “[Targeting unit]” represents a binding domain orbinding unit (and in particular an ISVD such as a Nanobody) that targetsthe polypeptide of the invention to a specific cell, tissue or organ

TABLE D-2b Schematic Representation of Some PD1 and/or LAG3 Binders ofthe Invention with a Half-Life Extending ISVD. [LAG3 binder of theinvention]-[HLE] [HLE]-[LAG3 binder of the invention] [LAG3 binder ofthe invention]-[HLE]-X(n) [HLE]-[LAG3 binder of the invention]-X(n)[LAG3 binder of the invention]-[LAG3 binder of the invention]-[HLE][LAG3 binder of the invention]-[HLE]-[LAG3 binder of the invention][HLE]-[LAG3 binder of the invention]-[LAG3 binder of the invention][LAG3 binder of the invention]-[LAG3 binder of the invention]-[HLE]-X(n) [LAG3 binder of the invention]-[HLE]-[LAG3 binder of theinvention]- X(n) [HLE]-[LAG3 binder of the invention]-[LAG3 binder ofthe invention]- X(n) [LAG3 binder of the invention]-[Other]-[HLE] [LAG3binder of the invention]-[LAG3 binder of the invention]-[Other]-[Other]-[HLE] [LAG3 binder of the invention]-[Other]-[HLE]-X(n) [LAG3binder of the invention]-[LAG3 binder of the invention]-[Other]-[Other]-[HLE]-X(n) [LAG3 binder of the invention]-[HLE]-[Other][HLE]-[LAG3 binder of the invention]-[Other] [HLE]-[Other]-[LAG3 binderof the invention] [Other]-[LAG3 binder of the invention]-[HLE][Other]-[HLE]-[LAG3 binder of the invention] [LAG3 binder of theinvention]-[Other]-[HLE]-X(n) [LAG3 binder of theinvention]-[HLE]-[Other]-X(n) [HLE]-[LAG3 binder of theinvention]-[Other]-X(n) [HLE]-[Other]-[LAG3 binder of theinvention]-X(n) [Other]-[LAG3 binder of the invention]-[HLE]-X(n)[Other]-[HLE]-[LAG3 binder of the invention]-X(n) [LAG3 binder of theinvention]-[Targeting unit]-[HLE] [LAG3 binder of theinvention]-[HLE]-[Targeting unit] [HLE]-[LAG3 binder of theinvention]-[Targeting unit] [Targeting unit]-[LAG3 binder of theinvention]-[HLE] [Targeting unit]-[HLE]-[LAG3 binder of the invention][HLE]-[Targeting unit]-[LAG3 binder of the invention] [LAG3 binder ofthe invention]-[Targeting unit]-[HLE]-X(n) [LAG3 binder of theinvention]-[HLE]-[Targeting unit]-X(n) [HLE]-[LAG3 binder of theinvention]-[Targeting unit]-X(n) [Targeting unit]-[LAG3 binder of theinvention]-[HLE]-X(n) [Targeting unit]-[HLE]-[LAG3 binder of theinvention]-X(n) [HLE]-[Targeting unit]-[LAG3 binder of theinvention]-X(n) [LAG3 binder of the invention]-[LAG3 binder of theinvention]- [Targeting unit]-[HLE] [LAG3 binder of the invention]-[LAG3binder of the invention]- [HLE]-[Targeting unit] [LAG3 binder of theinvention]-[HLE]-[LAG3 binder of the invention]-[Targeting unit][HLE]-[LAG3 binder of the invention]-[LAG3 binder of theinvention]-[Targeting unit] [LAG3 binder of the invention]-[PD-1binderof the invention]- [Targeting unit]-[HLE]-X(n) [LAG3 binder of theinvention]-[LAG3 binder of the invention]- [HLE]-[Targeting unit]-X(n)[LAG3 binder of the invention]-[HLE]-[LAG3 binder of theinvention]-[Targeting unit]-X(n) [HLE]-[LAG3 binder of theinvention]-[LAG3 binder of the invention]-[Targeting unit]-X(n)[Targeting unit]-[LAG3 binder of the invention]-[LAG3 binder of theinvention]-[HLE] [Targeting unit]-[LAG3 binder of the invention]-[HLE]-[LAG3 binder of the invention] [Targeting unit]-[HLE]-[LAG3 binder ofthe invention]- [LAG3 binder of the invention] [HLE]-[Targetingunit]-[LAG3 binder of the invention]- [LAG3 binder of the invention][Targeting unit]-[LAG3 binder of the invention]-[LAG3 binder of theinvention]-[HLE]-X(n) [Targeting unit]-[LAG3 binder of theinvention]-[HLE]- [LAG3 binder of the invention]-X(n) [Targetingunit]-[HLE]-[LAG3 binder of the invention]- [LAG3 binder of theinvention]-X(n) [HLE]-[Targeting unit]-[LAG3 binder of the invention]-[LAG3 binder of the invention]-X(n) [LAG3 binder of the invention]-[HLE][HLE]-[LAG3 binder of the invention] [LAG3 binder of theinvention]-[HLE]-X(n) [HLE]-[LAG3 binder of the invention]-X(n) [LAG3binder of the invention]-[LAG3 binder of the invention]-[HLE] [LAG3binder of the invention]-[HLE]-[LAG3 binder of the invention][HLE]-[LAG3 binder of the invention]-[LAG3 binder of the invention][LAG3 binder of the invention]-[LAG3 binder of the invention]-[HLE]-X(n) [LAG3 binder of the invention]-[HLE]-[LAG3 binder of theinvention]- X(n) [HLE]-[LAG3 binder of the invention]-[LAG3 binder ofthe invention]- X(n) [LAG3 binder of the invention]-[Other]-[HLE]Legend: “[LAG3 binder of the invention]” represents a LAG3 bindingdomain or moiety or unit such as the LAG3 ISVD the ISVD 11B09 (L11V,A14P, R41P, N43K, A62S, A74S, K83R, V89L) as described herein “—”represents either a direct covalent linkage or a suitable linker, suchas a 9GS, 15GS or 35GS linker “X(n)” represents a C-terminal extensionas defined herein such as a single alanine residue. “[HLE]” represents ahalf-life extending binding domain or binding unit (and in particular ahalf-life extending ISVD, such as a Nanobody), such as an ISVD (and inparticular Nanobody) against (human) serum albumin such as the anti-HSAISVD ALB11002 as described herein; “[Other]” represents a binding domainor binding unit (e.g., an ISVD such as a Nanobody) against an epitopethat is different from the PD1 binder, e.g., one or more CTLA4, BTLA,LAG3 and/or CD27 ISVDs, such as the PD1 ISVD 102C12 (E1D, L11V, A14P,A74S, K83R, I89L) as described herein “[Targeting unit]” represents abinding domain or binding unit (and in particular an ISVD such as aNanobody) that targets the polypeptide of the invention to a specificcell, tissue or organ

In an embodiment of the invention, the PD1/LAG3 binders of the presentinvention are as summarized below in Table D-3

TABLE D-3 PD1/LAG3 Binders of the Present Invention. DescriptionSequence Name: F023700899 EVQLVESGGGLVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: PGKEREFVA VITWSGGITYYADSVKGRFTISRDNSKNTVY1PD102C12 (A14P, A74S, K83R)-35GS- LQMNSLRPEDTAIYYCAG DKHQSSWYDYWGQGTLVTVSSG 1PD102C12 (A14P, A74S, K83R)-35GS-GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVEF0237611B09-35GS-F0237611B09-35GS- SGGGLVQPGGSLRLSCAASGSIAS IHAMGWERQAPGKERE ALB11002 FVA VITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLTarget: hPD-1/hLAG-3 RPEDTAIYYCAG DKHQSSWYDY WGQGTLVTVSSGGGGSGGSEQ ID NO: 106 GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFS DYVNGWERQARGNEREFVA AISESGGRTHYADAVKGRFTISRDNAKNTLYLQMNSLKPEDTA VYYCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFS DYVNG WERQARGNEREEVAAISESGGRTHYADAVKGRFTISRDNAKNTLYLQMNSLKPE DTAVYYCATT LLWWTSEYAPIKANDYDYWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL RPEDTAVYYCTIGGSLSRSSQGTLVTVSSName: F023700931 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 PGKEREFVA VITWSGGITYYADSVKGRFTISRDNSKNTVY(E1D, L11V, A14P, A74S ,K83R,  LQMNSLRPEDTALYYCAG DKHQSSWYDYWGQGTLVTVSSG I89L)-35GS-1PD102C12GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE (L11V, A14P, A74S, K83R, I89L)-SGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKERE 35GS-F0237611B09 FVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSL (L11V, A14P, R41P, N43K, A62S, RPEDTALYYCAG DKHQSSWYDY WGQGTLVTVSSGGGGSGGA74S, K83R, V89L)-35GS-F0237611B09GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVV (L11V, A14P, R41P, N43K, A62S, QPGGSLRLSCAASGRTF SDYVNG WERQAPGKEREFVA AISA74S, K83R, V89L)-35GS-ALB11002-AESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTA Target: hPD-1/hLAG-3 LYYCATTLLWWTSEYAPIKANDY DY WGQGTLVTVSSGGGG SEQ ID NO: 107SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGG GVVQPGGSLRLSCAASGRTFS DYVNGWERQAPGKEREEVA AISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPE DTALYYCATTLLWWTSEYAPIKANDY DY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL RPEDTALYYCTIGGSLSRSSQGTLVTVSSAName: F023701016 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQA Description:PGKEREFVA VITWSGGITYYADSVKGRFTISRDNSKNTVY102C12 (E1D, L11V, A14P, A74S,  LQMNSLRPEDTALYYCAG DKHQSSWYDYWGQGTLVTVSSG K83R, 189L)-20GS- GGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLS102C12 (L11V, A14P, A74S, K83R,  CAASGSIAS IHAMG WERQAPGKEREFVAVITWSGGITYYA I89L)-20GS- DSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDKHF0237611B09 (L11V, A14P, R41P,  QSSWYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEV N43K, A62S, A74S, K83R, V89L)-20GS-QLVESGGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGF0237611B09 (L11V, A14P, R41P,  KEREFVA AISESGGRTHYADSVKGRFTISRDNSKNTLYLQ N43K, A62S, A74S, K83R, V89L)-20GS-MNSLRPEDTALYYCAT TLLWWTSEYAPIKANDY DY WGQGTALB11002-A Target: hPD-1/hLAG-3 LVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPSEQ ID NO: 108 GGSLRLSCAASGRTFS DYVNG WERQAPGKEREFVA AISES GGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALY YCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVSSGGGGSG GGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSS QGTLVTVSSA Name: F023701017DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQA Description: PGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVY 102C12 (E1D, L11V, A14P, A74S, LQMNSLRPEDTALYYCAG DKHQSSWYDY WGQGTLVTVSSG K83R, 189L)-9GS-GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIAS IH102C12 (L11V, A14P, A74S, K83R,  AMG WERQAPGKEREFVAVITWSGGITYYADSVKGRFTISR I89L)-9GS- DNSKNTVYLQMNSLRPEDTALYYCAG DKHQSSWYDYWGQG F0237611B09 (L11V, A14P, R41P, TLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAAN43K, A62S, A74S, K83R, V89L)-9GS- SGRTFS DYVNG WERQAPGKEREFVAAISESGGRTHYADSV F0237611B09 (L11V, A14P, R41P, KGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAT TLLWWTN43K, A62S, A74S, K83R, V89L)-9GS- SEYAPIKANDY DYWGQGTLVTVSSGGGGSGGGSEVQLVES ALB11002-A Target: hPD-1/hLAG-3GGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREF SEQ ID NO: 109 VAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLR PEDTALYYCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVS SGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTL VTVSSA Name: F023700924DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQA Description: 1PD102C12PGKEREFVA VITWSGGITYYADSVKGRFTISRDNSKNTVY (E1D, L11V, A14P, A74S, K83R, LQMNSLRPEDTALYYCAG DKHQSSWYDY WGQGTLVTVSSG 189L)-35GS-F0237611B09GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE (L11V, A14P, R41P, N43K, A62S, SGGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREA74S, K83R, V89L)-35GS-ALB11002-A FVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSL Target: hPD-1/hLAG-3 RPEDTALYYCATTLLWWTSEYAPIKANDY DY WGQGTLVTV SEQ ID NO: 110SSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Name: F023700969DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQA Description: PGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVY 102C12 (E1D, L11V, A14P, A74S, LQMNSLRPEDTALYYCAG DKHQSSWYDY WGQGTLVTVSSG K83R, 189L)-20GS-GGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLS F0237611B09 (L11V, A14P, R41P, CAASGRTFSDYVNGWERQAPGKEREFVA AISESGGRTHYAN43K, A62S, A74S, K83R, V89L)-20GS-DSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAT TLLALB11002-A Target: hPD-1/hLAG-3 WWTSEYAPIKANDY DYWGQGTLVTVSSGGGGSGGGGSGGG SEQ ID NO: 111GSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTV SSA Name: F023700970DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQA Description: 102C12 PGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVY (E1D, L11V, A14P, A74S, K83R,LQMNSLRPEDTALYYCAG DKHQSSWYDY WGQGTLVTVSSG 189L)-9GS-F0237611B09GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFS DY(L11V, A14P, R41P, N43K, A62S,  VMG WFRQAPGKEREFVAAISESGGRTHYADSVKGRFTTSR A74S, K83R, V89L)-9GS-ALB11002-ADNSKNTLYLQMNSLRPEDTALYYCAT TLLWWTSEYAPIKA Target: hPD-1/hLAG-3 NDYD YWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPG SEQ ID NO: 112NSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYY CTIGGSLSRSSQGTLVTVSSAName: F023701163 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMGWFRQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVY A14P, W52aV, N73P, A74S, K83R,LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSG I89L,W100aF)-35GS-1PD102C12 GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE (L11V, A14P, W52aV, N73P, A74S,SGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKERE K83R, I89L, W100aF)-35GS- FVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSL F023700842-35GS- RPEDTALYYCAGDKHQSSFYDY WGQGTLVTVSSGGGGSGG F023700842-35GS-ALB11002-AGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVV Target: hPD-1/hLAG-3QPGGSLRLSCAASGRTFS DYVMG WERQAPGKEREFVA AIS SEQ ID NO: 113ESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTA LYYCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFS DYVMG WFRQAPGKEREEVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPE DTALYYCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL RPEDTALYYCTIGGSLSRSSQGTLVTVSSAName: F023701168 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WFRQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVY A14P, W52aV, N73P, A74S, K83R, LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGI89L, W100aF)-9GS-1PD102C12 (L11V,GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIAS IH A14P, W52aV, N73P, A74S, K83R,AMG WERQAPGKEREEVA VITVSGGITYYADSVKGRFTISRI89L, W100aF)-9GS-F023700842-9GS- DPSKNTVYLQMNSLRPEDTALYYCAG DKHQSSFYDYWGQG F023700842-9GS-ALB11002-A TLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAATarget: hPD-1/hLAG-3 SGRTFS DYVNG WERQAPGKEREFVA AISESGGRTHYADSVSEQ ID NO: 114 KGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAT TLLWWT SEYAPIKANDY DYWGQGTLVTVSSGGGGSGGGSEVQLVES GGGVVQPGGSLRLSCAASGRTFSDYVMGWFRQAPGKEREF VAA ISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLR PEDTALYYCAT TLLWWTSEYAPIKANDY DYWGQGTLVTVS SGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTL VTVSSA Name: F023701173DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WFRQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVY A14P, W52aV, N73P, A74S, K83R, LQMNSLRPEDTALYYCAG DKHQSSFYD YWGQGTLVTVSSGI89L, W100aF)-35GS-F023700842-35GS-GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE ALB11002-ASGGGVVQPGGSLRLSCAASGRTFS DYVMG WFRQAPGKERE Target: hPD-1/hLAG-3 FVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSL SEQ ID NO: 115 RPEDTALYYCATTLLWWTSEYAPIKANDYDY WGQGTLVTV SSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Name: F023701178DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVY A14P, W52aV, N73P, A74S, K83R, LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGI89L, W100aF)-9GS-F023700842-9GS- GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFSDY ALB11002-A VMG WFRQAPGKEREFVA AISESGGRTHYADSVKGRFTTSRTarget: hPD-1/hLAG-3 DNSKNTLYLQMNSLRPEDTALYYCAT TLLWWTSEYAPIKASEQ ID NO: 116 NDYDY WGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYY CTIGGSLSRSSQGTLVTVSSAName: F023701161 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVY A14P, W52aV, N73Q, A74S, K83R, LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGI89L, W100aF)-35GS-1PD102C12,  GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE(L11V, A14P, W52aV, N73Q, A74S,  SGGGVVQPGGSLRLSCAASGSIAS IHAMGWERQAPGKERE K83R, I89L, W100aF)-35GS- FVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSL F023700842-35GS- F023700842-35GS-RPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGGGGSGGALB11002-A Target: hPD-1/hLAG-3 GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVSEQ ID NO: 117 QPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREFVA AISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTA LYYCAT TLLWWTSEYAPIKANDYDYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREEVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPE DTALYYCAT TLLWWTSEYAPIKANDYDYWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL RPEDTALYYCTIGGSLSRSSQGTLVTVSSAName: F023701166 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVY A14P, W52aV, N73Q, A74S, K83R, I89L,LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGW100aF)-9GS-1PD102C12 (L11V, A14P,GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIAS IH W52aV, N73Q, A74S, K83R, I89L,AMG WERQAPGKEREFVA VITVSGGITYYADSVKGRFTISR W100aF)-9GS-F023700842-9GS-DQSKNTVYLQMNSLRPEDTALYYCAG DKHQSSFYDY WGQG F023700842-9GS-ALB11002-ATLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAA Target: hPD-1/hLAG-3SGRTFSDYVNGWERQAPGKEREFVA AISESGGRTHYADSV SEQ ID NO: 118KGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAT TLLWWT SEYAPIKANDYDYWGQGTLVTVSSGGGGSGGGSEVQLVES GGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREFVA AISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLR PEDTALYYCATTLLWWTSEYAPIKANDYDY WGQGTLVTVS SGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTL VTVSSA Name: F023701171DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVY A14P, W52aV, N73Q, A74S, K83R, I89L,LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSG W100aF)-35GS-F023700842-35GS-GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE ALB11002-ASGGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKERE Target: hPD-1/hLAG-3 FVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSL SEQ ID NO: 119 RPEDTALYYCATTLLWWTSEYAPIKANDYDY WGQGTLVTV SSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTESSEGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Name: F023701176DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVA VITVSGGITYYADSVKGRFTISRDQSKNTVY A14P, W52aV, N73Q, A74S, K83R, I89L, LQMNSLRPEDTALYYCAGDKHQSSFYDY WGQGTLVTVSSG W100aF)-9GS-F023700842-9GS-GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFS DY ALB11002-A VMG WFRQAPGKEREFVAAISESGGRTHYADSVKGRFTTSR Target: hPD-1/hLAG-3 DNSKNTLYLQMNSLRPEDTALYYCATTLLWWTSEYAPIKA SEQ ID NO: 120 NDYDY WGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTESSEGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYY CTIGGSLSRSSQGTLVTVSSAName: F023701162 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDSSKNTVY A14P, W52aV, N73S, A74S, K83R, I89L,LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGW100aF)-35GS-1PD102C1 (L11V, A14P,GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE W52aV, N73S, A74S, K83R, I89L,SGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQAPGKERE W100aF)-35GS-F023700842-35GS-FVA VITVSGGITYYADSVKGRFTISRDSSKNTVYLQMNSL F023700842-35GS-ALB11002-ARPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGGGGSGG Target: hPD-1/hLAG-3GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVV SEQ ID NO: 121QPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREFVA AISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTA LYYCAT TLLWWTSEYAPIKANDYDYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREEVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPE DTALYYCAT TLLWWTSEYAPIKANDYDYWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL RPEDTALYYCTIGGSLSRSSQGTLVTVSSAName: F023701167 DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDSSKNTVY A14P, W52aV, N73S, A74D, K83R, I89L,LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSGW100aF)-9GS-1PD102C12 (L11V, A14P,GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIAS IH W52aV, N73S, A74S, K83R, I89L,AMG WERQAPGKEREFVA VITVSGGITYYADSVKGRFTISR W100aF)-9GS-F023700842-9GS-DSSKNTVYLQMNSLRPEDTALYYCAG DKHQSSFYDY WGQG F023700842-9GS-ALB11002-ATLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAA Target: hPD-1/hLAG-3 SGRTFSDYVNG WERQAPGKEREFVA AISESGGRTHYADSV SEQ ID NO: 122KGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAT TLLWW T SEYAPIKANDYDYWGQGTLVTVSSGGGGSGGGSEVQLVES GGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKEREFVA AISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLR PEDTALYYCATTLLWWTSEYAPIKANDYDY WGQGTLVTVS SGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTL VTVSSA Name: F023701172DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDSSKNTVY A14P, W52aV, N73S, A74S, K83R, I89L,LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSG W100aF)-35GS-F023700842-35GS-GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE ALB11002-ASGGGVVQPGGSLRLSCAASGRTFS DYVNG WERQAPGKERE Target: hPD-1/hLAG-3 FVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSL SEQ ID NO: 123 RPEDTALYYCATTLLWWTSEYAPIKANDYDY WGQGTLVTV SSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Name: F023701177DVQLVESGGGVVQPGGSLRLSCAASGSIAS IHAMG WERQADescription: 1PD102C12 (E1D, L11V, PGKEREFVAVITVSGGITYYADSVKGRFTISRDSSKNTVY A14P, W52aV, N73S, A74S, K83R, I89L,LQMNSLRPEDTALYYCAG DKHQSSFYDY WGQGTLVTVSSG W100aF)-9GS-F023700842-9GS-GGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFS DY ALB11002-A VMG WFRQAPGKEREFVAAISESGGRTHYADSVKGRFTTSR Target: hPD-1/hLAG-3 DNSKNTLYLQMNSLRPEDTALYYCATTLLWWTSEYAPIKA SEQ ID NO: 124 NDYDY WGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTESSEGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYY CTIGGSLSRSSQGTLVTVSSA *PD1binder and LAG3 binder CDRs underscored and/or boldedOptionally, the first residue of any binder moiety in the molecule issubstituted with a D or an E as appropriate.

The present invention includes any PD1/LAG3 binder comprising the aminoacid sequence of SEQ ID NO: 106-124 or an amino acid sequence comprising80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99%) amino acidsequence identity (i.e., comparing the full amino acid sequences)wherein the PD1/LAG3 binder retains the ability to bind to PD1 and LAG3and, optionally, HSA.

The present invention includes any PD1/LAG3 binder having the followingarrangement of moieties:

-   -   PD1 binder 1PD102C12 (A14P,A74S,K83R)-    -   Peptide linker, e.g., 35GS-    -   PD1 binder 1PD102C12 (A14P,A74S,K83R)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F0237611B09-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F0237611B09-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002        or    -   PD1 binder 1PD102C12 (E1D,L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 35GS-    -   PD1 binder 1PD102C12 (L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 102C12 (E1D,L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 20GS-    -   PD1 binder 102C12(L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 20GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 20GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 20GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 102C12 (E1D,L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 9GS-    -   PD1 binder 102C12 (L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D,L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 102C12(E1D,L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 20GS-    -   LAG3 binder        F0237611B09(L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 20GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 102C12 (E1D,L11V,A14P,A74S,K83R,I89L)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F0237611B09        (L11V,A14P,R41P,N43K,A62S,A74S,K83R,V89L)-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73P, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 35GS-    -   PD1 binder 1PD102C12 (L11V, A14P, W52aV, N73P, A74S, K83R, I89L,        W100aF)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73P, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 9GS-    -   PD1 binder 1PD102C12 (L11V, A14P, W52aV, N73P, A74S, K83R, I89L,        W100aF)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73P, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73P, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73Q, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 35GS-    -   PD1 binder 1PD102C12 (L11V, A14P, W52aV, N73Q, A74S, K83R, I89L,        W100aF)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73Q, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 9GS-    -   PD1 binder 1PD102C12 (L11V, A14P, W52aV, N73Q, A74S, K83R, I89L,        W100aF)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73Q, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73Q, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73S, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 35GS-    -   PD1 binder 1PD102C12 (L11V, A14P, W52aV, N73S, A74S, K83R, I89L,        W100aF)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73S, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 9GS-    -   PD1 binder 1PD102C12 (L11V, A14P, W52aV, N73S, A74S, K83R, I89L,        W100aF)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73S, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 35GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 35GS    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine        or    -   PD1 binder 1PD102C12 (E1D, L11V, A14P, W52aV, N73S, A74S, K83R,        I89L, W100aF)-    -   Peptide linker, e.g., 9GS-    -   LAG3 binder F023700842-    -   Peptide linker, e.g., 9GS-    -   Human serum albumin binder, e.g., ALB11002-    -   Optional C-terminal extension alanine.

In an embodiment of the invention, the PD1/LAG3 binders of the presentinvention include the following arrangement of moieties:

-   -   PD1 binder SEQ ID NO: 98;    -   35GS linker SEQ ID NO: 58;    -   PD1 binder SEQ ID NO: 98;    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 63;    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 63;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 57;    -   35GS linker SEQ ID NO: 58;    -   PD1 binder SEQ ID NO: 99;    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 57;    -   20GS linker SEQ ID NO: 100;    -   PD1 binder SEQ ID NO: 99;    -   20GS linker SEQ ID NO: 100;    -   LAG3 binder SEQ ID NO: 64;    -   20GS linker SEQ ID NO: 100;    -   LAG3 binder SEQ ID NO: 64;    -   20GS linker SEQ ID NO: 100;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 57;    -   9GS linker SEQ ID NO: 125;    -   PD1 binder SEQ ID NO: 99;    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 57;    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 57;    -   20GS linker SEQ ID NO: 100;    -   LAG3 binder SEQ ID NO: 64;    -   20GS linker SEQ ID NO: 100;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 57;    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 105;    -   35GS linker SEQ ID NO: 58;    -   PD1 binder SEQ ID NO: 105 (D1E);    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 105;    -   9GS linker SEQ ID NO: 125;    -   PD1 binder SEQ ID NO: 105 (D1E);    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 105;    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 105;    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 104;    -   35GS linker SEQ ID NO: 58;    -   PD1 binder SEQ ID NO: 104 (D1E);    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 104;    -   9GS linker SEQ ID NO: 125;    -   PD1 binder SEQ ID NO: 104 (D1E);    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 104;    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 104;    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 103 (N73S);    -   35GS linker SEQ ID NO: 58;    -   PD1 binder SEQ ID NO:103 (D1E, N73S);    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 103 (N73S);    -   9GS linker SEQ ID NO: 125;    -   PD1 binder SEQ ID NO:103 (D1E, N73S);    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 103 (N73S);    -   35GS linker SEQ ID NO: 58;    -   LAG3 binder SEQ ID NO: 64;    -   35GS linker SEQ ID NO: 58;    -   HSA binder SEQ ID NO: 59;    -   Alanine        or    -   PD1 binder SEQ ID NO: 103 (N73S);    -   9GS linker SEQ ID NO: 125;    -   LAG3 binder SEQ ID NO: 64;    -   9GS linker SEQ ID NO: 125;    -   HSA binder SEQ ID NO: 59;    -   Alanine

Cross-Blocking Antibodies

The present invention also provides cross-blocking binders that are ableto cross-block binding of any of the binders disclosed herein (e.g.,F023700899; F023700931; F023701016; F023701017; F023700924; F023700969;F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8;F023701161; F023701166; F023701171; F023701176; F023701162; F023701167;F023701172; or F023701177). Such cross-blocking binders may be anymolecule that exhibits such cross-blocking, e.g., an ISVD, Nanobody,antibody or antigen-binding fragment thereof.

In general, a binder (e.g., ISVD such as Nanobody) or antibody orantigen-binding fragment thereof that “cross-blocks” a reference binderor “cross competes with” a reference binder refers to a binder (e.g.,ISVD such as Nanobody) or antibody or antigen-binding fragment thereofthat blocks binding of the reference binder to its antigen in acompetition assay by 50% or more, and conversely, the reference binderblocks binding of the binder (e.g., ISVD such as Nanobody) or antibodyor antigen-binding fragment thereof to its antigen in a competitionassay by 50% or more. Cross-blocking or cross-competition can bedetermined any assay known in the art, including suface plasmonresonance (SPR), ELISA and flow cytometry.

In an embodiment of the invention, cross-blocking is determined by useof a Biacore assay. For convenience reference is made to two binders,the scope of the present invention includes antibodies and antigenbinding fragments thereof, e.g., Fab fragments, that cross-block abinder of the present invention. A Biacore machine (for example theBiacore 3000) is operated in line with the manufacturer'srecommendations.

Thus, in one cross-blocking assay, PD1 or LAG3 is coupled to a CMSBiacore chip using standard amine coupling chemistry to generate a PD1or LAG3-coated surface. For example, 200-800 resonance units of PD1 orLAG3 would be coupled to the chip (or any amount that gives easilymeasurable levels of binding but that is readily saturable by theconcentrations of test reagent being used).

The two binders (termed A* and B*) to be assessed for their ability tocross-block each other are mixed at a one to one molar ratio of bindingsites in a suitable buffer to create the test mixture.

The concentration of each binder in the test mix should be high enoughto readily saturate the binding sites for that binder on the PD1 or LAG3molecules captured on the Biacore chip. The binders in the mixture areat the same molar concentration.

Separate solutions containing binder A* alone and binder B* alone arealso prepared. Binder A* and binder B* in these solutions should be inthe same buffer and at the same concentration as in the test mix.

The test mixture is passed over the PD1 or LAG3-coated Biacore chip andthe total amount of binding recorded. The chip is then treated in such away as to remove the bound binders without damaging the chip-bound PD1or LAG3. In an embodiment of the invention, this is done by treating thechip with 30 mM HCl for 60 seconds.

The solution of binder A* alone is then passed over the PD1 orLAG3-coated surface and the amount of binding recorded. The chip isagain treated to remove all of the bound binder without damaging thechip-bound PD1 or LAG3.

The solution of binder B* alone is then passed over the PD1 orLAG3-coated surface and the amount of binding recorded.

The maximum theoretical binding of the mixture of binder A* and binderB* is next calculated, and is the sum of the binding of each binder whenpassed over the PD1 or LAG3 surface alone. If the actual recordedbinding of the mixture is less than this theoretical maximum, then thetwo binders are cross-blocking each other.

Thus, in general, a cross-blocking binder according to the invention isone which will bind to PD1 or LAG3 in the above Biacore cross-blockingassay such that, during the assay and in the presence of a secondbinder, the recorded binding is between, for example, 80% and 0.1%(e.g., 80% to 4%) of the maximum theoretical binding, for examplebetween 75% and 0.1% (e.g., 75% to 4%) of the maximum theoreticalbinding, for example, between 70% and 0.1% (e.g., 70% to 4%) of maximumtheoretical binding (as just defined above) of the two binders incombination.

In an embodiment of the invention, an ELISA assay is used fordetermining whether a PD1 and/or LAG3 binder cross-blocks or is capableof cross-blocking according to the invention.

The general principal of the assay is to have an PD1 or LAG3 bindercoated onto the wells of an ELISA plate. An excess amount of a second,potentially cross-blocking, anti-PD1 or LAG3 binder is added in solution(i.e., not bound to the ELISA plate). A limited amount of PD1 or LAG3 isthen added to the wells. The coated binder and the binder in solutioncompete for binding of the limited number of PD1 or LAG3 molecules. Theplate is washed to remove PD1 or LAG3 that has not been bound by thecoated binder and to also remove the second, solution phase binder aswell as any complexes formed between the second, solution phase binderand PD1 or LAG3. The amount of bound PD1 or LAG3 is then measured usingan appropriate PD1 or LAG3 detection reagent. A binder in solution thatis able to cross-block the coated binder will be able to cause adecrease in the number of PD1 or LAG3 molecules that the coated bindercan bind relative to the number of PD1 or LAG3 molecules that the coatedbinder can bind in the absence of the second, solution phase, binder.

Expression Methods

The present invention includes recombinant methods for making PD1 and/orLAG3 binders (e.g., an ISVD such as a Nanobody) of the present invention(e.g., F023700899; F023700931; F023701016; F023701017; F023700924;F023700969; F023700970; F023701163; F023701168; F023701173; F02370117F023701176; 8; F023701161; F023701166; F023701171; F023701176;F023701162; F023701167; F023701172; or F023701177) comprising (i)introducing a polynucleotide encoding the amino acid sequence of saidPD1 and/or LAG3 binder, for example, wherein the polynucleotide is in avector and/or is operably linked to a promoter; (ii) culturing the hostcell (e.g., CHO or Pichia or Pichia pastoris) under condition favorableto expression of the polynucleotide and, (iii) optionally, isolating thePD1 and/or LAG3 binder from the host cell and/or medium in which thehost cell is grown. See e.g., WO 04/041862, WO 2006/122786, WO2008/020079, WO 2008/142164 or WO 2009/068627.

The invention also relates to polynucleotides that encode PD1 and/orLAG3 binders of the present invention (e.g., an ISVD such as a Nanobody)as described herein (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177). Thepolynucleotides may, in an embodiment of the invention, be operablylinked to one or more control sequences. The polynucleotide may be inthe form of a plasmid or vector. Again, such polynucleotides can begenerally as described in the published patent applications of AblynxN.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079,WO 2008/142164 or WO 2009/068627.

The invention also relates to hosts or host cells that contain suchpolynucleotides encoding PD1 and/or LAG3 binders, vectors, and/or PD1and/or LAG3 binder polypeptide described herein (e.g., F023700899;F023700931; F023701016; F023701017; F023700924; F023700969; F023700970;F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161;F023701166; F023701171; F023701176; F023701162; F023701167; F023701172;or F023701177). Again, such host cells can be generally as described inthe published patent applications of Ablynx N.V., such as for example WO04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO2009/068627. Examples of specific host cells are discussed below.

Eukaryotic and prokaryotic host cells, including mammalian cells ashosts for expression of the PD1 and/or LAG3 binder (e.g., an ISVD suchas a Nanobody) are well known in the art and include many immortalizedcell lines available from the American Type Culture Collection (ATCC).These include, inter alfa, Chinese hamster ovary (CHO) cells, NSO, SP2cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells(COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells,3T3 cells, HEK-293 cells and a number of other cell lines. Mammalianhost cells include human, mouse, rat, dog, monkey, pig, goat, bovine,horse and hamster cells. Cell lines of particular preference areselected through determining which cell lines have high expressionlevels. Other cell lines that may be used are insect cell lines (e.g.,Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterialcells, plant cells and fungal cells. Fungal cells include yeast andfilamentous fungus cells including, for example, Pichia pastoris, Pichiafinlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri),Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichiaguercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichiasp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha,Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei,Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusariumvenenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candidaalbicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurosporacrassa. The present invention includes any host cell (e.g., a CHO cellor Pichia cell, e.g., Pichia pastoris) containing an PD1 and/or LAG3binder of the present invention (e.g., F023700899; F023700931;F023701016; F023701017; F023700924; F023700969; F023700970; F023701163;F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166;F023701171; F023701176; F023701162; F023701167; F023701172; orF023701177) or containing a polynucleotide encoding such a binder orcontaining a vector that contains the polynucleotide.

Further, expression of a PD1 and/or LAG3 binder (e.g., an ISVD such as aNanobody) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase gene expressionsystem (the GS system) is a common approach for enhancing expressionunder certain conditions. The GS system is discussed in whole or part inconnection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997and European Patent Application No. 89303964.4. Thus, in an embodimentof the invention, the mammalian host cells (e.g., CHO) lack a glutaminesynthetase gene and are grown in the absence of glutamine in the mediumwherein, however, the polynucleotide encoding the immunoglobulin chaincomprises a glutamine synthetase gene which complements the lack of thegene in the host cell. Such host cells containing the binder orpolynucleotide or vector as discussed herein as well as expressionmethods, as discussed herein, for making the binder using such a hostcell are part of the present invention.

The present invention includes methods for purifying a PD1 and/or LAG3binder (e.g., an ISVD such as a Nanobody) (e.g., F023700899; F023700931;F023701016; F023701017; F023700924; F023700969; F023700970; F023701163;F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166;F023701171; F023701176; F023701162; F023701167; F023701172; orF023701177) comprising introducing a sample (e.g., culture medium, celllysate or cell lysate fraction, e.g., a soluble fraction of the lysate)comprising the PD1 and/or LAG3 binder to a purification medium (e.g.,cation-exchange medium, anion-exchange medium and/or hydrophobicexchange medium) and either collecting purified PD1 and/or LAG3 binderfrom the flow-through fraction of said sample that does not bind to themedium; or, discarding the flow-through fraction and eluting bound PD1and/or LAG3 binder from the medium and collecting the eluate. In anembodiment of the invention, the medium is in a column to which thesample is applied. In an embodiment of the invention, the purificationmethod is conducted following recombinant expression of the antibody orfragment in a host cell, e.g., wherein the host cell is first lysed and,optionally, the lysate is purified of insoluble materials prior topurification on a medium; or wherein the PD1 and/or LAG3 binder issecreted into the culture medium by the host cell and the medium or afraction thereof is applied to the purification medium.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal. Therefore, the particular glycosylation pattern of a PD1 and/orLAG3 binder (e.g., an ISVD such as a Nanobody) (e.g., F023700899;F023700931; F023701016; F023701017; F023700924; F023700969; F023700970;F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161;F023701166; F023701171; F023701176; F023701162; F023701167; F023701172;or F023701177) will depend on the particular cell line or transgenicanimal used to produce the PD1 and/or LAG3 binder. PD1 and/or LAG3binders comprising only non-fucosylated N-glycans are part of thepresent invention and may be advantageous, because non-fucosylatedantibodies have been shown to typically exhibit more potent efficacythan their fucosylated counterparts both in vitro and in vivo (See forexample, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S.Pat. Nos. 6,946,292 and 7,214,775). These PD1 and/or LAG3 binders withnon-fucosylated N-glycans are not likely to be immunogenic because theircarbohydrate structures are a normal component of the population thatexists in human serum IgG.

The present invention includes PD1 and/or LAG3 binders (e.g., an ISVDsuch as a Nanobody) comprising N-linked glycans that are typically addedto immunoglobulins produced in Chinese hamster ovary cells (CHO N-linkedglycans) or to engineered yeast cells (engineered yeast N-linkedglycans), such as, for example, Pichia pastoris. For example, in anembodiment of the invention, the PD1 and/or LAG3 binder comprises one ormore of the “engineered yeast N-linked glycans” or “CHO N-linkedglycans” that are set forth in FIG. 4 (e.g., G0 and/or G0-F and/or G1and/or G1-F and/or and/or G2-F and/or Man5). In an embodiment of theinvention, the PD1 and/or LAG3 binder comprises the engineered yeastN-linked glycans, i.e., G0 and/or G1 and/or G2, optionally, furtherincluding Man5. In an embodiment of the invention, the PD1 and/or LAG3binders comprise the CHO N-linked glycans, i.e., G0-F, G1-F and G2-F,optionally, further including G0 and/or G1 and/or G2 and/or Man5. In anembodiment of the invention, about 80% to about 95% (e.g., about 80-90%,about 85%, about 90% or about 95%) of all N-linked glycans on the PD1and/or LAG3 binders are engineered yeast N-linked glycans or CHON-linked glycans. See Nett et al. Yeast. 28(3): 237-252 (2011); Hamiltonet al. Science. 313(5792): 1441-1443 (2006); Hamilton et al. Curr OpinBiotechnol. 18(5): 387-392 (2007). For example, in an embodiment of theinvention, an engineered yeast cell is GFI5.0 or YGLY8316 or strains setforth in U.S. Pat. No. 7,795,002 or Zha et al. Methods Mol Biol.988:31-43 (2013). See also international patent application publicationno. WO2013/066765.

Combinations

In particular embodiments, the PD1 and/or LAG3 binders (e.g., an ISVDsuch as a Nanobody) of the present invention (e.g., F023700899;F023700931; F023701016; F023701017; F023700924; F023700969; F023700970;F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161;F023701166; F023701171; F023701176; F023701162; F023701167; F023701172;or F023701177) may be used alone, or in association with other, furthertherapeutic agents and/or therapeutic procedures, for treating orpreventing any disease such as cancer, e.g., as discussed herein, in asubject in need of such treatment or prevention. Compositions or kits,e.g., pharmaceutical compositions comprising a pharmaceuticallyacceptable carrier, comprising such PD1 and/or LAG3 binders inassociation with further therapeutic agents are also part of the presentinvention.

The term “in association with” indicates that the components, a PD1and/or LAG3 binder (e.g., an ISVD such as a Nanobody) of the presentinvention (e.g., F023700899; F023700931; F023701016; F023701017;F023700924; F023700969; F023700970; F023701163; F023701168; F023701173;F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176;F023701162; F023701167; F023701172; or F023701177) along with anotheragent such as pembrolizumab or nivolumab, can be formulated into asingle composition, e.g., for simultaneous delivery, or formulatedseparately into two or more compositions (e.g., a kit). Each componentcan be administered to a subject at a different time than when the othercomponent is administered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at intervals overa given period of time. Moreover, the separate components may beadministered to a subject by the same or by a different route (e.g.,wherein a PD1 and/or LAG3 binder of the present invention isadministered parenterally and paclitaxel is administered orally).

In particular embodiments, the PD1 and/or LAG3 binders (e.g., an ISVDsuch as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) may beused in association with an anti-cancer therapeutic agent orimmunomodulatory drug such as an immunomodulatory receptor inhibitor,e.g., an antibody or antigen-binding fragment thereof that specificallybinds to the receptor.

In an embodiment of the invention, a PD1 and/or LAG3 binder (e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) is inassociation with one or more of an inhibitors (e.g., a small organicmolecule or an antibody or antigen-binding fragment thereof) such as: anMTOR (mammalian target of rapamycin) inhibitor, a cytotoxic agent, aplatinum agent a BRAF inhibitor, a CDK4/6 inhibitor an EGFR inhibitor, aVEGF inhibitor, a microtubule stabilizer, a taxane, a CD20 inhibitor, aCD52 inhibitor, a CD30 inhibitor, a RANK (Receptor activator of nuclearfactor kappa-B) inhibitor, a RANKL (Receptor activator of nuclear factorkappa-B ligand) inhibitor, an ERK inhibitor, a MAP Kinase inhibitor, anAKT inhibitor, a MEK inhibitor, a PI3K inhibitor, a HER1 inhibitor, aHER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, a Bc12 inhibitor, aCD22 inhibitor, a CD79b inhibitor, an ErbB2 inhibitor, or a farnesylprotein transferase inhibitor.

In an embodiment of the invention, a PD1 and/or LAG3 binder(e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) is inassociation with one or more of: anti-PD1 antibody or antigen-bindingfragment thereof (e.g., pembrolizumab, nivolumab, CT-011), anti-PDL1,anti-CTLA4, anti-TIM3, anti-CS1, (e.g., elotuzumab), anti-KIR2DL1/2/3(e.g., lirilumab), anti-CD27, anti-CD137 (e.g., urelumab), anti-GITR(e.g., TRX518), anti-PD-L1 (e.g., BMS-936559, MSB0010718C or MPDL3280A),anti-PD-L2, anti-ILT1, anti-ILT2, anti-ILT3, anti-ILT4, anti-ILT5,anti-ILT6, anti-ILT7, anti-ILT8, anti-CD40, anti-0X40, anti-CD137,anti-KIR2DL1, anti-KIR2DL2/3, anti-KIR2DL4, anti-KIR2DL5A,anti-KIR2DL5B, anti-KIR3DL1, anti-KIR3DL2, anti-KIR3DL3, anti-NKG2A,anti-NKG2C, anti-NKG2E, or any small organic molecule inhibitor of suchtargets; IL-10, anti-IL10, anti-TSLP (thymic stromal lymphopoietin) orPEGylated IL-10.

In an embodiment of the invention, the molecular weight of thepolyethylene glycol (PEG) moiety, on a PEGylated IL-10 molecule, isabout 12,000 daltons or about 20,000 daltons. In an embodiment of theinvention, PEGylated IL-10 (e.g., PEGylated human IL-10) comprises oneor more polyethylene glycol molecules covalently attached via a linker(e.g., C₂₋₁₂ alkyl such as —CH₂CH₂CH₂—) to a single amino acid residueof a single subunit of IL-10, wherein said amino acid residue is thealpha amino group of the N-terminal amino acid residue or the epsilonamino group of a lysine residue. In an embodiment of the inventionPEGylated IL-10 is: (PEG)_(b)-L-NH-IL-10; wherein b is 1-9 and L is aC₂₋₁₂ alkyl linker moiety covalently attached to a nitrogen (N) of thesingle amino acid residue of the IL-10. In an embodiment of theinvention, the IL-10 of PEGylated IL-10 has the formula:[X—O(CH₂CH₂O)_(n)]_(b)-L-NH-IL-10, wherein X is H or C₁₋₄ alkyl; n is 20to 2300; b is 1 to 9; and L is a C₁₋₁₁ alkyl linker moiety which iscovalently attached to the nitrogen (N) of the alpha amino group at theamino terminus of one IL-10 subunit; provided that when b is greaterthan 1, the total of n does not exceed 2300. See U.S. Pat. No.7,052,686.

In an embodiment of the invention, the anti-IL-10 antibody orantigen-binding fragment thereof (e.g., humanized antibody) comprisesthe CDRs set forth below:

CDR-L1: (SEQ ID NO: 71) KTSQNIFENLA  CDR-L2: (SEQ ID NO: 72) NASPLQA CDR-L3: (SEQ ID NO: 73) HQYYSGYT  CDR-H1: (SEQ ID NO: 74) GFTFSDYHMA CDR-H2: (SEQ ID NO: 75) SITLDATYTYYRDSVRG  CDR-H3: (SEQ ID NO: 76)HRGFSVVAILDY (See US7,662,379)

In an embodiment of the invention, the anti-TSLP antibody orantigen-binding fragment thereof (e.g., humanized antibody) comprisesthe CDRs set forth below:

CDR-H1:  (SEQ ID NO: 77) GYIFTDYAMH  CDR-H2:  (SEQ ID NO: 78)TFIPLLDTSDYNQNFK  CDR-H3:  (SEQ ID NO: 79) MGVTHSYVMDA  CDR-L1: (SEQ ID NO: 80) RASQPISISVH  CDR-L2:  (SEQ ID NO: 81) FASQSIS  CDR-L3: (SEQ ID NO: 82) QQTFSLPYT  (see WO2008/76321)

In an embodiment of the invention, the anti-CD27 antibody orantigen-binding fragment thereof (e.g., humanized antibody) comprisesthe CDRs set forth below:

CDR-H1: (SEQ ID NO: 83) GEIIKATYMH  CDR-H2: (SEQ ID NO: 84)RIDPANGETKYDPKFQV  CDR-H3: (SEQ ID NO: 85) YAWYFDV  CDR-L1:(SEQ ID NO: 86) RASENIYSFLA  CDR-L2: (SEQ ID NO: 87) HAKTLAE  CDR-L3:(SEQ ID NO: 88) QHYYGSPLT  (See WO2012/04367).

Thus, the present invention includes compositions comprising a PD1and/or LAG3 binder (e.g., an ISVD such as a Nanobody) (e.g., F023700899;F023700931; F023701016; F023701017; F023700924; F023700969; F023700970;F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161;F023701166; F023701171; F023701176; F023701162; F023701167; F023701172;or F023701177) in association with pembrolizumab; as well as methods fortreating or preventing cancer in a subject comprising administering aneffective amount of the PD1 and/or LAG3 binder in association withpembrolizumab (e.g., pembrolizumab dosed at 200 mg once every threeweeks) to the subject. Optionally, the subject is also administered inassociation with a another further therapeutic agent.

In an embodiment of the invention, a PD1 and/or LAG3 binder (e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) is inassociation with a pembrolizumab antibody which comprises animmunoglobulin heavy chain (or CDR-H1, CDR-H2 and CDR-H3 thereof)comprising the amino acid sequence:

(SEQ ID NO: 89) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;and an immunoglobulin light chain (or CDR-L1, CDR-L2 and CDR-L3 thereof)comprising the amino acid sequence:

(SEQ ID NO: 90) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLTYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTEGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In an embodiment of the invention, a PD1 and/or LAG3 binder (e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) is inassociation with an antibody comprising an immunoglobulin heavy chain(or CDR-H1, CDR-H2 and CDR-H3 thereof) comprising the amino acidsequence:

(SEQ ID NO: 91) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;and an immunoglobulin light chain (or CDR-L1, CDR-L2 and CDR-L3 thereof)comprising the amino acid sequence:

(SEQ ID NO: 92) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In an embodiment of the invention, a PD1 and/or LAG3 binder (e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) is inassociation with any one or more of: 13-cis-retinoic acid,3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone,4-hydroxytamoxifen, 5-deooxyuridine, 5′-deoxy-5-fluorouridine,5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654,abirateroneacetate, abraxane, ABT-578, acolbifene, ADS-100380,aflibercept, ALT-110, altretamine, amifostine, aminoglutethimide,amrubicin, amsacrine, anagrelide, anastrozole, angiostatin, AP-23573,ARQ-197, arzoxifene, AS-252424, AS-605240, asparaginase, ATI3387,AT-9263, atrasentan, axitinib, AZD1152, Bacillus Calmette-Guerin (BCG)vaccine, batabulin, BC-210, besodutox, bevacizumab, BGJ398,bicalutamide, Bio111, BI0140, BKM120, bleomycin, BMS-214662, BMS-247550,BMS-275291, BMS-310705, bortezimib, buserelin, busulfan, calcitriol,camptothecin, canertinib, capecitabine, carboplatin, carmustine, CC8490,CEA (recombinant vaccinia-carcinoembryonic antigen vaccine), cediranib,CG-1521, CG-781, chlamydocin, chlorambucil, chlorotoxin, cilengitide,cimitidine, cisplatin, cladribine, clodronate, cobimetnib, COL-3,CP-724714, cyclophosphamide, cyproterone, cyproteroneacetate,cytarabine, cytosinearabinoside, dabrafenib, dacarbazine, dacinostat,dactinomycin, dalotuzumab, danusertib, dasatanib, daunorubicin,decatanib, deguelin, denileukin, deoxycoformycin, depsipeptide,diarylpropionitrile, diethylstilbestrol, diftitox, DNE03, docetaxel,dovitinib, doxorubicin, droloxifene, edotecarin, yttrium-90labeled-edotreotide, edotreotide, EKB-569, EMD121974, encorafenib,endostatin, enzalutamide, enzastaurin, epirubicin, epithilone B,ERA-923, erbitux, erlotinib, estradiol, estramustine, etoposide,everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol,floxuridine, fludarabine, fludrocortisone, fluoxymesterone, flutamide,FOLFOX regimen, fulvestrant, galeterone, ganetespib, gefitinib,gemcitabine, gimatecan, glucopyranosyl lipid A, goserelin, goserelinacetate, gossypol, GSK461364, GSK690693, EMIR-3339,hydroxyprogesteronecaproate, hydroxyurea, IC87114, idarubicin,idoxyfene, ifosfamide, IM862, imatinib, IMC-1C11, imiquimod, INC280,INCB24360, IN01001, interferon, interleukin-2, interleukin-12,ipilimumab, irinotecan, JNJ-16241199, ketoconazole, KRX-0402, lapatinib,lasofoxifene, LEE011, letrozole, leucovorin, leuprolide, leuprolideacetate, levamisole, liposome entrapped paclitaxel, lomustine,lonafarnib, lucanthone, LY292223, LY292696, LY293646, LY293684,LY294002, LY317615, LY3009120, marimastat, mechlorethamine,medroxyprogesteroneacetate, megestrolacetate, MEK162, melphalan,mercaptopurine, mesna, methotrexate, mithramycin, mitomycin, mitotane,mitoxantrone, a suspension of heat killed Mycobacterium obuense,tozasertib, MLN8054, natitoclax, neovastat, Neratinib, neuradiab,nilotinib, nilutimide, nolatrexed, NVP-BEZ235, oblimersen, octreotide,ofatumumab, oregovomab, ornatuzumab, orteronel, oxaliplatin, paclitaxel,palbociclib, pamidronate, panitumumab, pazopanib, PD0325901, PD184352,PEG-interferon, pemetrexed, pentostatin, perifosine,phenylalaninemustard, PI-103, pictilisib, PIK-75, pipendoxifene,PKI-166, plicamycin, poly-ICLC, porfimer, prednisone, procarbazine,progestins, PSK protein bound polysaccharide (derived from Basidiomycetecoriolus versicolor), PLX8394, PX-866, R-763, raloxifene, raltitrexed,razoxin, ridaforolimus, rituximab, romidepsin, RTA744, rubitecan,scriptaid, Sdx102, seliciclib, selumetinib, semaxanib, SF1126,sirolimus, SN36093, sorafenib, spironolactone, squalamine, SR13668,streptozocin, SU6668, suberoylanalide hydroxamic acid, sunitinib,synthetic estrogen, talampanel, talimogene laherparepvec, tamoxifen,temozolomide, temsirolimus, teniposide, tesmilifene, testosterone,tetrandrine, TGX-221, thalidomide, 6-thioguanine, thiotepa, ticilimumab,tipifarnib, tivozanib, TKI-258, TLK286, TNF□ (tumor necrosis factoralpha), topotecan, toremifene citrate, trabectedin, trametinib,trastuzumab, tretinoin, trichostatin A, triciribinephosphatemonohydrate, triptorelin pamoate, TSE-424, uracil mustard, valproicacid, valrubicin, vandetanib, vatalanib, VEGF trap, vemurafenib,vinblastine, vincristine, vindesine, vinorelbine, vitaxin, vitespan,vorinostat, VX-745, wortmannin, Xr311, Z-100 hot water extract ofBacillus tuberculosis, zanolimumab, ZK186619, ZK-304709, ZM336372 orZSTK474.

In an embodiment of the invention, a PD1 and/or LAG3 binder (e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) is inassociation with one or more antiemetics including, but not limited to:casopitant (GlaxoSmithKline), Netupitant (MGI-Helsinn) and other NK-1receptor antagonists, palonosetron (sold as Aloxi by MGI Pharma),aprepitant (sold as Emend by Merck and Co.; Rahway, N.J.),diphenhydramine (sold as Benadryl® by Pfizer; New York, N.Y.),hydroxyzine (sold as Atarax® by Pfizer; New York, N.Y.), metoclopramide(sold as Reglan® by AH Robins Co,; Richmond, Va.), lorazepam (sold asAtivan® by Wyeth; Madison, N.J.), alprazolam (sold as Xanax® by Pfizer;New York, N.Y.), haloperidol (sold as Haldol® by Ortho-McNeil; Raritan,N.J.), droperidol (Inapsine®), dronabinol (sold as Marinol® by SolvayPharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (sold as Decadron®by Merck and Co.; Rahway, N.J.), methylprednisolone (sold as Medrol® byPfizer; New York, N.Y.), prochlorperazine (sold as Compazine® byGlaxosmithkline; Research Triangle Park, N.C.), granisetron (sold asKytril® by Hoffmann-La Roche Inc.; Nutley, N.J.), ondansetron (sold asZofran® by by Glaxosmithkline; Research Triangle Park, N.C.), dolasetron(sold as Anzemet® by Sanofi-Aventis; New York, N.Y.), tropisetron (soldas Navoban® by Novartis; East Hanover, N.J.).

Other side effects of cancer treatment include red and white blood celldeficiency. Accordingly, in an embodiment of the invention, a PD1 and/orLAG3 binder (e.g., an ISVD such as a Nanobody) (e.g., F023700899;F023700931; F023701016; F023701017; F023700924; F023700969; F023700970;F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161;F023701166; F023701171; F023701176; F023701162; F023701167; F023701172;or F023701177) is in association with an agent which treats or preventssuch a deficiency, such as, e.g., filgrastim, PEG-filgrastim,erythropoietin, epoetin alfa or darbepoetin alfa.

In an embodiment of the invention, a PD1 and/or LAG3 binder (e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) is inassociation with a vaccine. In an embodiment of the invention, thevaccine is an anti-cancer vaccine, a peptide vaccine or a DNA vaccine.For example, in an embodiment of the invention, the vaccine is a tumorcell (e.g., an irradiated tumor cell) or a dendritic cell (e.g., adendritic cell pulsed with a tumor peptide).

In an embodiment of the invention, a PD1 and/or LAG3 binder (e.g., anISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) isadministered in association with a therapeutic procedure. A therapeuticprocedure is one or more steps carried out by a physician or clinicianin treating a subject which is intended to alleviate one or moresymptoms (e.g., of cancer and/or infectious disease) in the treatedsubject, whether by inducing the regression or elimination of suchsymptoms or by inhibiting the progression of such symptom(s), e.g.,cancer symptoms such as tumor growth or metastasis, by any clinicallymeasurable degree.

In an embodiment of the invention, a therapeutic procedure isanti-cancer radiation therapy. For example, in an embodiment of theinvention, the radiation therapy is external beam therapy (EBT): amethod for delivering a beam of high-energy X-rays to the location ofthe tumor. The beam is generated outside the patient (e.g., by a linearaccelerator) and is targeted at the tumor site. These X-rays can destroythe cancer cells and careful treatment planning allows the surroundingnormal tissues to be spared. No radioactive sources are placed insidethe patient's body. In an embodiment of the invention, the radiationtherapy is proton beam therapy: a type of conformal therapy thatbombards the diseased tissue with protons instead of X-rays. In anembodiment of the invention, the radiation therapy is conformal externalbeam radiation therapy: a procedure that uses advanced technology totailor the radiation therapy to an individual's body structures.

In an embodiment of the invention, the radiation therapy isbrachytherapy: the temporary placement of radioactive materials withinthe body, usually employed to give an extra dose—or boost—of radiationto an area.

In an embodiment of the invention, a surgical procedure administered inassociation with a PD1 and/or LAG3 binder (e.g., F023700899; F023700931;F023701016; F023701017; F023700924; F023700969; F023700970; F023701163;F023701168; F023701173; F02370117 F023701176; 8; F023701161; F023701166;F023701171; F023701176; F023701162; F023701167; F023701172; orF023701177) is surgical tumorectomy.

Therapeutic Uses

The invention includes a method for the prevention and/or treatment ofat least one disease or disorder that can be prevented or treated by theuse of a PD1 and/or LAG3 binder (e.g., an ISVD such as a Nanobody)(e.g., F023700899; F023700931; F023701016; F023701017; F023700924;F023700969; F023700970; F023701163; F023701168; F023701173; F02370117F023701176; 8; F023701161; F023701166; F023701171; F023701176;F023701162; F023701167; F023701172; or F023701177) of the presentinvention, optionally in association with a further therapeutic agent ortherapeutic procedure, which method comprises administering, to asubject in need thereof, a pharmaceutically active amount of the PD1and/or LAG3 binder, and/or of a pharmaceutical composition comprisingthe same.

“Treat” or “treating” means to administer PD1 and/or LAG3 binders (e.g.,an ISVD such as a Nanobody) of the present invention (e.g., F023700899;F023700931; F023701016; F023701017; F023700924; F023700969; F023700970;F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161;F023701166; F023701171; F023701176; F023701162; F023701167; F023701172;or F023701177), to a subject having one or more symptoms of a diseasefor which the PD1 and/or LAG3 binders are effective, e.g., in thetreatment of a subject having cancer or an infectious disease, or beingsuspected of having cancer or infectious disease, for which the agenthas therapeutic activity. Typically, the PD1 and/or LAG3 binder isadministered in an “effective amount” or “effective dose” which willalleviate one or more symptoms (e.g., of cancer or infectious disease)in the treated subject or population, whether by inducing the regressionor elimination of such symptoms or by inhibiting the progression of suchsymptom(s), e.g., cancer symptoms such as tumor growth or metastasis, byany clinically measurable degree. The effective amount of the PD1 and/orLAG3 binder may vary according to factors such as the disease stage,age, and weight of the patient, and the ability of the drug to elicit adesired response in the subject.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk from, the diseases anddisorders mentioned herein. Generally, the treatment regimen will befollowed until the desired therapeutic effect is achieved and/or for aslong as the desired therapeutic effect is to be maintained. Again, thiscan be determined by the clinician.

The invention also relates to a pharmaceutical composition thatcomprises at least one amino acid sequence, a PD1 and/or LAG3 binder(e.g., an ISVD such as a Nanobody), polypeptide or compound as describedherein, such as F023700899; F023700931; F023701016; F023701017;F023700924; F023700969; F023700970; F023701163; F023701168; F023701173;F02370117 F023701176; 8; F023701161; F023701166; F023701171; F023701176;F023701162; F023701167; F023701172; or F023701177, and optionally atleast one pharmaceutically acceptable carrier, diluent or excipient.Such preparations, carriers, excipients and diluents may generally be asdescribed in the published patent applications of Ablynx N.V., such asfor example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164or WO 2009/068627.

To prepare pharmaceutical or sterile compositions of the PD1 and/or LAG3binders (e.g., an ISVD such as a Nanobody) of the present invention(e.g., F023700899; F023700931; F023701016; F023701017; F023700924;F023700969; F023700970; F023701163; F023701168; F023701173; F02370117F023701176; 8; F023701161; F023701166; F023701171; F023701176;F023701162; F023701167; F023701172; or F023701177), the PD1 and/or LAG3binders is admixed with a pharmaceutically acceptable carrier orexcipient. See, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa.(1984). Such compositions are part of the present invention.

The scope of the present invention includes dessicated, e.g.,freeze-dried, compositions comprising an PD1 and/or LAG3 binders (e.g.,an ISVD such as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) or apharmaceutical composition thereof that includes a pharmaceuticallyacceptable carrier but substantially lacks water.

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency and/or the half-life of the specificfusion proteins or constructs to be used, the specific route ofadministration and the specific pharmaceutical formulation orcomposition used, the Nanobodies and polypeptides of the invention willgenerally be administered in an amount between 1 gram and 0.01 microgramper kg body weight per day, preferably between 0.1 gram and 0.1microgram per kg body weight per day, such as about 1, 10, 100 or 1000microgram per kg body weight per day, either continuously (e.g., byinfusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, for example on the basis of the factors cited above andhis expert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, half-life and similar factors well known to the skilledperson.

The mode of administration of a PD1 and/or LAG3 binder (e.g., an ISVDsuch as a Nanobody) (e.g., F023700899; F023700931; F023701016;F023701017; F023700924; F023700969; F023700970; F023701163; F023701168;F023701173; F02370117 F023701176; 8; F023701161; F023701166; F023701171;F023701176; F023701162; F023701167; F023701172; or F023701177) to asubject can vary. Routes of administration include oral, rectal,transmucosal, intestinal, parenteral; intramuscular, subcutaneous,intradermal, intramedullary, intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, intraocular, inhalation,insufflation, topical, cutaneous, transdermal, or intra-arterial.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, in determining the dose, the dose begins with anamount somewhat less than the optimum dose and it is increased by smallincrements thereafter until the desired or optimum effect is achievedrelative to any negative side effects. Important diagnostic measuresinclude those of symptoms of, e.g., the inflammation or level ofinflammatory cytokines produced. In general, it is desirable that abiologic that will be used is derived from the same species as theanimal targeted for treatment, thereby minimizing any immune response tothe reagent. In the case of human subjects, for example, chimeric,humanized and fully human antibodies are may be desirable. Guidance inselecting appropriate doses of PD1 and/or LAG3 binders (e.g., F023700924or F023700931) is available (see, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003)New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al.(2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J.Med. 343:1594-1602).

Whether a disease symptom has been alleviated can be assessed by anyclinical measurement typically used by physicians or other skilledhealthcare providers to assess the severity or progression status ofthat symptom. While an embodiment of the present invention (e.g., atreatment method or article of manufacture) may not be effective inalleviating the target disease symptom(s) in every subject, it shouldalleviate the target disease symptom(s) in a statistically significantnumber of subjects as determined by any statistical test known in theart such as the Student's t-test, the chi²-test, the U-test according toMann and Whitney, the Kruskal-Wallis test (H-test),Jonckheere-Terpstra-test and the Wilcoxon-test.

As the PD1 and/or LAG3 binders of the present invention (e.g.,F023700899; F023700931; F023701016; F023701017; F023700924; F023700969;F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8;F023701161; F023701166; F023701171; F023701176; F023701162; F023701167;F023701172; or F023701177) are capable of binding to PD1 (e.g., an ISVDsuch as a Nanobody), they can in particular be used for treatment orprevention of cancer, metastatic cancer, a solid tumor, a hematologiccancer, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma,neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer,bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breastcancer, prostate cancer, bone cancer, lung cancer, non-small cell lungcancer, gastric cancer, colorectal cancer, cervical cancer, synovialsarcoma, head and neck cancer, squamous cell carcinoma, multiplemyeloma, renal cell cancer, retinoblastoma, hepatoblastoma,hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney,Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma,pituitary adenoma, vestibular schwannoma, a primitive neuroectodermaltumor, medulloblastoma, astrocytoma, anaplastic astrocytoma,oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemiavera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma,thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer,breast cancer and gastric cancer.

PD1 and/or LAG3 binders (e.g., an ISVD such as a Nanobody) of thepresent invention can be used for treatment or prevention of infectiousdiseases such as, for example, viral infection, bacterial infection,fungal infection or parasitic infection. In an embodiment of theinvention, the viral infection is infection with a virus selected fromthe group consisting of human immunodeficiency virus (HIV), ebola virus,hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6,HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus orarboviral encephalitis virus. In an embodiment of the invention, thebacterial infection is infection with a bacteria selected from the groupconsisting of Chlamydia, rickettsial bacteria, mycobacteria,staphylococci, streptococci, pneumonococci, meningococci and gonococci,klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacteriumdiphtherias, Salmonella, bacilli, Vibrio cholerae, Clostridium tetan,Clostridium botulinum, Bacillus anthricis, Yersinia pestis,Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella. In anembodiment of the invention, the fungal infection is infection with afungus selected from the group consisting of Candida (albicans, krusei,glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus(fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioidesbrasiliensis, Coccidioides immitis and Histoplasma capsulatum. In anembodiment of the invention, the parasitic infection is infection with aparasite selected from the group consisting of Entamoeba histolytica,Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia Zambia,Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

The present invention also includes methods for:

-   -   inhibiting LAG3 binding to MHC class II molecules;    -   competing with MHC class II molecules for LAG3 binding;    -   binding a LAG3 binder, e.g., PD1/LAG3 binder, to native LAG3 on        the surface of activated CD4+ and/or CD8+ T-cells;    -   inhibiting LAG3 homodimerization; and/or    -   stimulating antigen-specific T-cell production of IL-2        in the body of a subject by administering the PD1/LAG3 binder        (e.g., F023700899; F023700931; F023701016; F023701017;        F023700924; F023700969; F023700970; F023701163; F023701168;        F023701173; F02370117 F023701176; 8; F023701161; F023701166;        F023701171; F023701176; F023701162; F023701167; F023701172; or        F023701177) to the subject; or in vitro by contacting LAG3 with        the PD1/LAG3 binder. Such activities can be mediated via the        LAG3 binder. Thus, such methods may also be performed with any        binder that includes a LAG3 binder.

The present invention also includes methods for:

-   -   blocking binding between PD1 and PD-L1 and/or PD-L2    -   binding a PD1 binder, e.g., a PD1/LAG3 binder, to B-cells and/or        T-cells    -   blocking PD1 mediated T-cell inhibition, T-cell apoptosis and/or        T-cell exhaustion in the body of a subject by administering the        PD1/LAG3 binder (e.g., F023700924 or F023700931) to the subject;        or in vitro by contacting PD1 with the PD1/LAG3 binder. Such        activities can be mediated via the PD1 binder. Thus, such        methods may also be performed with any binder that includes a        PD1 binder.

The present invention further includes methods for increasing thehalf-life of a binder, such as a PD1 and/or:LAG3 binder by fusing thebinder to an anti-human serum albumin binder such as ALB11002.

The invention also relates to methods of treatment of the aforementioneddiseases and disorders, which generally comprise administering to asubject in need thereof (i.e. suffering from one of the aforementioneddiseases) a therapeutically effective amount of a PD1 and/or LAG3 binder(e.g., an ISVD such as a Nanobody) of the invention (e.g., F023700899;F023700931; F023701016; F023701017; F023700924; F023700969; F023700970;F023701163; F023701168; F023701173; F02370117 F023701176; 8; F023701161;F023701166; F023701171; F023701176; F023701162; F023701167; F023701172;or F023701177). The invention also relates to a PD1 and/or LAG3 binderof the invention for use in the prevention or treatment of one of theaforementioned diseases or disorders.

The PD1 and/or LAG3 binders (e.g., an ISVD such as a Nanobody) (e.g.,F023700899; F023700931; F023701016; F023701017; F023700924; F023700969;F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8;F023701161; F023701166; F023701171; F023701176; F023701162; F023701167;F023701172; or F023701177), polypeptides, compounds, and polynucleotides(e.g., vectors) described herein are preferably administered to thecirculation. As such, they can be administered in any suitable mannerthat allows the PD1 and/or LAG3 binders, polypeptides, compounds, andpolynucleotides to enter the circulation, such as intravenously, viainjection or infusion, or in any other suitable manner (including oraladministration, subcutaneous administration, intramuscularadministration, administration through the skin, intranasaladministration, administration via the lungs, etc.) that allows the PD1and/or LAG3 binders, polypeptides, compounds, and polynucleotides toenter the circulation. Suitable methods and routes of administrationwill be clear to the skilled person, again for example also from theteaching of the published patent applications of Ablynx N.V., such asfor example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164or WO 2009/068627.

The present invention also provides an injection device comprising anyof the PD1 and/or LAG3 binders (e.g., an ISVD such as a Nanobody) (e.g.,F023700899; F023700931; F023701016; F023701017; F023700924; F023700969;F023700970; F023701163; F023701168; F023701173; F02370117 F023701176; 8;F023701161; F023701166; F023701171; F023701176; F023701162; F023701167;F023701172; or F023701177), polypeptides or polynucleotides set forthherein or a pharmaceutical composition thereof. An injection device is adevice that introduces a substance into the body of a patient via aparenteral route, e.g., intramuscular, subcutaneous or intravenous. Forexample, an injection device may be a syringe (e.g., pre-filled with thepharmaceutical composition, such as an auto-injector) which, forexample, includes a cylinder or barrel for holding fluid to be injected(e.g., comprising the PD1 and/or LAG3 binder or a pharmaceuticalcomposition thereof), a needle for piecing skin and/or blood vessels forinjection of the fluid; and a plunger for pushing the fluid out of thecylinder and through the needle bore. In an embodiment of the invention,an injection device that comprises an PD1 and/or LAG3 binder or apharmaceutical composition thereof is an intravenous (IV) injectiondevice. Such a device includes the PD1 and/or LAG3 binder or apharmaceutical composition thereof in a cannula or trocar/needle whichmay be attached to a tube which may be attached to a bag or reservoirfor holding fluid (e.g., saline; or lactated ringer solution comprisingNaCl, sodium lactate, KCl, CaCl₂ and optionally including glucose)introduced into the body of the subject through the cannula ortrocar/needle. The PD1 and/or LAG3 binder or a pharmaceuticalcomposition thereof may, in an embodiment of the invention, beintroduced into the device once the trocar and cannula are inserted intothe vein of a subject and the trocar is removed from the insertedcannula. The IV device may, for example, be inserted into a peripheralvein (e.g., in the hand or arm); the superior vena cava or inferior venacava, or within the right atrium of the heart (e.g., a central IV); orinto a subclavian, internal jugular, or a femoral vein and, for example,advanced toward the heart until it reaches the superior vena cava orright atrium (e.g., a central venous line). In an embodiment of theinvention, an injection device is an autoinjector; a jet injector or anexternal infusion pump. A jet injector uses a high-pressure narrow jetof liquid which penetrate the epidermis to introduce the PD1 and/or LAG3binder or a pharmaceutical composition thereof to a patient's body.External infusion pumps are medical devices that deliver the PD1 and/orLAG3 binder or a pharmaceutical composition thereof into a patient'sbody in controlled amounts. External infusion pumps may be poweredelectrically or mechanically. Different pumps operate in different ways,for example, a syringe pump holds fluid in the reservoir of a syringe,and a moveable piston controls fluid delivery, an elastomeric pump holdsfluid in a stretchable balloon reservoir, and pressure from the elasticwalls of the balloon drives fluid delivery. In a peristaltic pump, a setof rollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

It should also be noted that the Figures, any Sequence Listing and theExperimental Part/Examples are only given to further illustrate theinvention and should not be interpreted or construed as limiting thescope of the invention and/or of the appended claims in any way, unlessexplicitly indicated otherwise herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

EXAMPLES

These examples are intended to exemplify the present invention are not alimitation thereof. Compositions and methods set forth in the Examplesform part of the present invention.

Example 1 Monovalent Human PD-1 Nanobody Binding to CHO.Hpd-1

Binding to cell-expressed human PD-1 was evaluated on human PD-1over-expressing CHO cells. A Nanobody dilution series was prepared inassay buffer: PBS/10% FBS/0.05% sodium azide. 1×10⁵ cells/well weretransferred to a 96-well V-bottom plate and resuspended in 100 μLNanobody dilution. After 30 minutes incubation at 4° C., the cells werewashed with 100 μL/well assay buffer and resuspended in 100 μL/well of 1μg/ml anti-FLAG (Sigma, F1804) or anti-HIS (AbD Serotec, MCA 1396).Samples were incubated for 30 minutes at 4° C., washed with 100 μL/wellassay buffer, and resuspended in 100 μL/well of 5 μg/ml PE-labeled Goatanti-mouse IgG (Jackson ImmunoResearch, 115-116-071). Samples wereincubated for 30 minutes at 4° C., washed, and resuspended in 100μL/well of 5 nM TOPRO3 (LifeTechnologies, T3606) solution beforeanalysis on FACS CANTO II (BD). The data from these experiments are setforth in FIG. 5.

This Example demonstrated that the anti-human PD-1 monovalent NanobodyF023700706 bound to human PD-1 in a manner similar to the F023700275monovalent Nanobody from which it was derived.

Example 2 Monovalent Human LAG-3 Nanobody Binding to 3A9.hLAG-3

Binding to cell-expressed human LAG-3 was evaluated on human LAG-3over-expressing 3A9 cells. A Nanobody dilution series was prepared inassay buffer: PBS/10% FBS/0.05% sodium azide. 1×10⁵ cells/well weretransferred to a 96-well V-bottom plate and resuspended in 100 μLNanobody dilution. After 30 minutes incubation at 4° C., the cells werewashed with 100 μL/well assay buffer and resuspended in 100 μL/well of 1μg/ml anti-FLAG (Sigma, F1804). Samples were incubated for 30 minutes at4° C., washed with 100 μL/well assay buffer and resuspended in 100μL/well of 5 μg/ml PE-labeled Goat anti-mouse IgG (JacksonImmunoResearch, 115-116-071). Samples were incubated for 30 minutes at4° C., washed, and resuspended in 100 μL/well of 5 nM TOPRO3(LifeTechnologies, T3606) solution before analysis on FACS CANTO II(BD). The data from these experiments are set forth in FIG. 6.

This Example demonstrated that the sequence optimized anti-human LAG-3monovalent Nanobody F023700842 bound to human LAG-3 in a manner similarto the original F02376611B09 monovalent Nanobody from which it wasderived.

Example 3 Human PD-1 Nanobody-Containing Multispecific Nanobody Bindingto CHO.hPD-1 and 3A9.rhesusPD-1

Binding to cell-expressed human PD-1 and rhesus PD-1 was evaluated onhuman PD-1 over-expressing CHO cells and rhesus PD-1 over-expressing 3A9cells, respectively. A Nanobody dilution series was prepared in assaybuffer: PBS/10% FBS/0.05% sodium azide. 1×10⁵ cells/well weretransferred to a 96-well V-bottom plate and resuspended in 100 μLNanobody dilution. After 30 minutes incubation at 4° C., the cells werewashed with 100 μL/well assay buffer and resuspended in 100 μL/well of 3μg/ml ABH0074, a monoclonal antibody that recognizes the albumin bindingNanobody half-life extension moiety. Samples were incubated for 30minutes at 4° C., washed with 100 μL/well assay buffer, and resuspendedin 100 μL/well of 5 μg/ml PE-labeled Goat anti-mouse IgG (JacksonImmunoResearch, 115-116-071). Samples were incubated for 30 minutes at4° C., washed and resuspended in 100 μL/well of 5 nM TOPRO3(LifeTechnologies, T3606) solution before analysis on FACS CANTO II(BD). The data from these experiments were set forth in FIG. 7(A-B).

This Example demonstrated that the bispecific anti-human PD-1/LAG-3Nanobodies F023700931 and F023700924 bound to both human PD 1 and rhesusPD-1. The monospecific, bivalent controls anti-human PD-1 F023700933 andanti-human LAG-3 F023700962 were also shown.

Example 4 Human LAG-3 Nanobody-Containing Multispecific Nanobody Bindingto CHO.hLAG3 and CHO.rhesus/cynoLAG3

Binding to cell-expressed human and non-human primate LAG-3 (theextracellular domain is identical between cynomolgus and rhesus monkey;therefore, the gene will be referred to a rhesus/cynoLAG-3) wasevaluated on human and rhesus/cynoLAG-3 over-expressing CHO cells. ANanobody dilution series was prepared in assay buffer: PBS/10% FBS/0.05%sodium azide. 1×10⁵ cells/well were transferred to a 96-well V-bottomplate and resuspended in 100 μL Nanobody dilution. After 30 minutesincubation at 4° C., the cells were washed with 100 μL/well assay bufferand resuspended in 100 μL/well of 1-3 μg/ml ABH0074, a monoclonalantibody that recognizes the albumin binding Nanobody half-lifeextension moiety. Samples were incubated for 30 minutes at 4° C., washedwith 100 μL/well assay buffer, and resuspended in 100 μL/well of 5 μg/mlPE-labeled Goat anti-mouse IgG (Jackson ImmunoResearch, 115-116-071).Samples were incubated for 30 minutes at 4° C., washed and resuspendedin 100 μL/well of 5 nM TOPRO3 (LifeTechnologies, T3606) solution beforeanalysis on FACSArray (BD). The data from these experiments are setforth in FIG. 8(A-B).

This Example demonstrated that the bispecific anti-human PD-1/LAG-3Nanobodies F023700931 and F023700924 bound to both human LAG-3 andrhesus/cynomolgous monkey LAG-3. The monospecific, bivalent controlsanti-human PD-1 F023700933 and anti-human LAG-3 F023700962 were alsoshown.

Example 5 Biophysical PD-L1-Fc and PD-L2-Fc Blockade Assay

Ligand competition assays were performed on human PD-1 over-expressingCHO cells. A Nanobody dilution series was prepared in assay buffer:PBS/10% FBS/0.05% sodium azide. Five-fold serial dilutions of Nanobodywere made starting from 1 μM (2×) and pre-diluted with 11 nM (2×) humanPD-L1-hFc or 29 nM (2×) human PD-L2-hFc in a total volume of 220 μL.2×10⁴ cells/well were seeded in V-bottom 96-well plates and resuspendedin 200 μL/well of the Nanobody/ligand dilution. After 90 minutesincubation at 4° C., the cells were washed with 100 μL/well assay bufferand resuspended in 100 μL/well PE-labeled Goat anti-human IgG (SouthernBiotech, 2043-09). Samples were incubated for 30 minutes at 4° C.,washed, and resuspended in 100 μL/well of 5 nM TOPRO3 (LifeTechnologies,T3606) solution before analysis on FACS CANTO II (BD). The data fromthese experiments are set forth in FIG. 9(A-H).

This Example demonstrated that the anti-human PD-1 Nanobody monovalentmodule F023700706 bound to human PD-1 and fully blocked its interactionwith PD-L1 (FIG. 9(A)) and PD-L2 (FIG. 9(B)) similar to the originalF023700275 monovalent module from which it was derived. In addition,this example demonstrated that the bispecific anti-human PD-1/LAG-3Nanobodies F023700931 and F023700924 bound to human PD-1 and fullyblocked their interaction with PD-L1 (FIG. 9(C)) and PD-L2 (FIG. 9(D)).The sequence optimized, monospecific, bivalent controls, anti human PD-1F023700933 and anti-human LAG-3 F023700962 were also shown (FIG.9(E-F)). Lastly, this example demonstrated that additional amino acidvariants of anti-human PD-1 Nanobody monovalent module F023700929 boundto human PD-1 and fully blocked its interaction with PD-L1 (FIG. 9(G))and PD-L2 (FIG. 9(H)). It should be noted that F023700706 and F023700929were the identical anti-human PD-1 monovalent Nanobody entity either asa FLAG3-HIS6 fusion protein or as a HIS6 fusion protein, respectively.

Example 6 Biophysical LAG-3-Fc Blockade Assay

Ligand competition assays were performed on human Daudi cells whichshowed high endogenous expression of surface major histocompatibilitycomplex (MHC) class II (ClassII). A Nanobody dilution series wasprepared in assay buffer: HBSS/2% FBS. Prior to the experiment, Fcreceptors on the Daudi cells were blocked with human Fc block (BDPharmingen, 564220) and 5 μg/mL goat IgG (Jackson ImmunoResearch,005-000-003) for 30 minutes at 4° C. Five-fold serial dilutions ofNanobody were made starting from 2 μM (2×) and pre-diluted with 80 nM(2×) human LAG-3-hFc in a total volume of 120 μL. 1×10⁵ cells/well wereseeded in V-bottom 96-well plates and resuspended in 100 μL/well of theNanobody/ligand dilution. After 30 minutes incubation at 4° C., thecells were washed and resuspended in 100 μL/well PE-labeled Goatanti-human IgG (Southern Biotech, 2043-09). Samples were incubated for15 minutes at 4° C., washed, and resuspended in 100 μL/well of 5 nMTOPRO3 (LifeTechnologies, T3606) solution before analysis on FACS CANTOII (BD). The data from these experiments are set forth in FIG. 10(A-C).

This Example demonstrated that the anti-human LAG-3 Nanobody monovalentmodule F023700842 bound to human LAG-3 and fully blocked its interactionwith MHC Class II similar to the original F0237611B09 monovalentNanobody from which it was derived (FIG. 10(A)). In addition, thisexample demonstrated that the bispecific anti human PD-1/LAG-3Nanobodies F023700931 and F023700924 bound to human LAG-3 and fullyblocked its interaction with MHC Class II (FIG. 10(B-C)). Themonospecific, bivalent controls anti-human PD-1 F023700933 and antihuman LAG-3 F023700962 were also shown.

Example 7 Proximity Dimerization Assay

A proximity dimerization assay was used that was based on abeta-Galactosidase Enzyme Fragment Complementation assay system. Theassay utilizes a fusion protein generated with a tag termed ProLink (PK)and a complementation protein in which the enzyme acceptor protein (EA)is fused to a second protein. U2OS cells were stably transfected withthe extracellular domain of LAG-3 (1-477) fused to the EA subunit andthe extracellular domain of PD-1 (1-199) fused to the PK subunit.U2OS.LAG3(1-477)-EA.PD1(1-199)-PK cell line #9 was plated inquadruplicate at 5,000 cells/well in DiscoverX CP5 plating media on384-well plates. Cells were allowed to attach for 4 hours at 37° C. in a5% carbon dioxide incubator. An 11 point, 1:3 dilution series ofNanobody samples were then added to the cells and incubated overnight(16 hours) at 37° C. in a 5% carbon dioxide incubator. PathHunterdetection reagent was added to the wells, incubated one hour at roomtemperature in the dark, and the plate was then read on an Envisionluminometer. The data from these experiments are set forth in FIG.11(A-B).

This Example demonstrated that the bispecific anti-human PD-1/LAG-3Nanobody F023700931 simultaneously bound to human PD-1 and human LAG-3expressed in the cell membrane due to bringing the PK and the EAsubunits together to form an active enzyme complex to give a lightsignal (FIG. 11(A)). The monospecific, bivalent controls anti-human PD-1F023700933 individually and the anti-human LAG-3 F023700962 individuallywere unable to bind both targets simultaneously and therefore did notgenerate a light signal. The individual modules in the bispecificanti-human PD-1/LAG-3 Nanobody F023700931 were linked with aGlycine-Serine linker that is 35 amino acids long (35GS). The bispecificanti-human PD-1/LAG-3 Nanobody F023701016 and F023701017 contained theidentical PD-1 and LAG-3 modules as F023700931 but the Glycine-Serinelinker between all modules in the constructs were 20 amino acids long(20GS) or 9 amino acids long (9GS), respectively. This example showedthat shortening the Glycine-Serine linker from 35GS to 9GS had minimalimpact on the ability to bind both targets simulataneously on the cellmembrane and give a light signal. Collectively, these data showed thatthe bispecific anti humanPD-1/LAG-3 Nanobody F023700931 can bind bothtargets simultaneously, if both targets were expressed by the same cell.

This Example also demonstrated that the bispecific anti-human PD-1/LAG-3Nanobody F023700924 simultaneously bound to human PD-1 and human LAG-3expressed in the cell membrane (FIG. 11(B)). The individual modules inthe bispecific anti-human PD-1/LAG-3 Nanobody F023700924 are linked witha Glycine-Serine linker that is 35 amino acids long (35GS). Thebispecific anti-human PD-1/LAG-3 Nanobody F023700969 and F023700970contained the identical PD-1 and LAG-3 modules as F023700924 but theGlycine-Serine linker between all modules in the construct were 20 aminoacids long (20GS) or 9 amino acids long (9GS), respectively. Thisexample showed that shortening the Glycine-Serine linker from 35GS to9GS had minimal impact on the ability to bind both targetssimulataneously and give a light signal. Collectively, these data showedthat the bispecific anti-human PD-1/LAG-3 Nanobody F023700924 could bindboth targets simultaneously, if both targets were expressed by the samecell.

Example 8 Engineered Jurkat.hPD-1.IL2luc+THP-1.PD-L1 Assay

Clone DT999A1 is a PD-1 transgene expressing Jurkat cell clone with anIL-2 mediated luciferase reporter (Jurkat.hPD-1.IL2luc).Jurkat.hPD-1.IL2luc were grown in RPMI media (Corning Cellgro10-040-CV)+heat inactivated 10% FBS (Hyclone SH30910.03)+2 mML-glutamine (Cellgro 25-005-CI)+2 ug/ml puromycin (Sigma P9620)+0.5mg/ml Geneticin (Gibco 10131-027). Cells were split twice per week afterseeding cells at 2×10⁵ cells/ml and were split when the density exceeded1×10⁶ cells/ml. PD-L1 transgene expressing THP-1 cells (THP-1.PD-L1)were grown in RPMI media +heat inactivated 10% FBS+2 mM L-glutamine+0.5ug/ml puromycin. Cells were split twice per week after seeding at3×10⁵cells/ml and were split when they reach 1×10⁶cells/ml.

The bioassay was setup using Assay Media (Phenol red free RPMI media(Gibco 11835-030)+10% dialyzed FBS (Hyclone, SH30079.03). Human albumin(Sigma, A8763) was prepared as 600 uM stock. A 4× (120 uM) solution wasprepared using assay media and 25 uL added to white walled tissueculture treated plates (Costar 3903). Dilute Nanobodies using assaymedia to get a starting 4× concentration. Make six 10-fold serialdilutions of the Nanobodies. Add 25 ul of the Nanobody titration to thewhite walled tissue culture treated plate containing albumin. Incubatethe Nanobodies+albumin for 20-30 minutes at room temperature. Harvest aT-75 flask of THP-1.PD-L1 cells, spin the cells, and resuspend in 10 mlof assay media. Count the cell suspension and adjust to get a cellsuspension of 4×10⁶cells/ml. Harvest a T-75 flask of Jurkat.hPD-1.IL2luccells, centrifuge cells, and resuspend in 10 ml of assay media. Countthe cell suspension and adjust to get Jurkat.hPD-1.IL2luc cellssuspension of 1×10⁶ cells/ml. Mix equal volume of THP-1.PD-L1cells+Jurkat.hPD-1.IL2luc cells. To this mixed suspension, add 2 ng/mlLPS (2×) and 100 ng/ml IFN-g (2×). Add 50 uL cell suspension containingthe stimulation conditions [IFN-g (R&D systems 285-IF/CF)+LPS (SigmaL4391)] to the Nanobody titration. Incubate the cells with the Nanobodytitration for approximately 22 hours in an incubator. At the end of 22hours, carefully add 10 ul of 55 ng/ml anti-CD3 antibody (BD Pharmingen555336; 11× working solution) and leave in the incubator for anadditional two hours. At the end of two hours incubation, stick whitetape (Perkin Elmer 6005199) to the bottom of the plate and add 100 ul ofOne-Glo reagent (Promega E6120). Incubate with shaking at roomtemperature for 3 minutes. Read the plate on a plate reader capable ofreading luminescence with an integration time of 0.1 secs. Raw data inRLU (Relative light units) can be plotted directly or plotted as foldchange in luciferase signal obtained by dividing the treatment wellswith wells that have no Nanobody. The data from these experiments areset forth in FIG. 12(A-E).

This Example demonstrated that the anti-human PD-1 Nanobody monovalentmodule, F023700706, bound to human PD-1 expressed by the T-cell line,blocks PD-1's interaction with PD-L1 expressed by the co-culture cellline to relieve the suppression that PD-L1 was providing to the T-cell;thereby, allowing the T-cell to respond, to a greater degree, to theT-cell receptor agonist based on inhibiting the PD-L1-mediatedsuppression (FIG. 12(A)). Furthermore, this Example demonstrated thatthe bispecific anti human PD-1/LAG-3 Nanobodies F023700931 andF023700924 bound to human PD-1 expressed by the T-cell line, blocksPD-1's interaction with PD-L1; thereby, allowing the T-cell to respondto a greater degree to the T-cell receptor agonist based on inhibitingthe PD-L1-mediated suppression (FIG. 12(B)). The monospecific, bivalentcontrols anti human PD-1 F023700933 and anti-human LAG-3 F023700962 werealso shown. Additionally, this Example showed that the bispecificanti-human PD-1/LAG-3 Nanobody F023700969 and F023700970 containing 20GSor 9GS linkers, respectively, had similiar potency as the parentF023700924 molecule containing 35GS linkers (FIG. 12(C)). Similarly, thebispecific anti-human PD-1/LAG-3 Nanobody F023701016 and F023701017containing 20GS or 9GS linkers, respectively, had similiar potency asthe parent F023700931 molecule containing 35GS linkers (FIG. 12(D)).Also, this Example demonstrated that additional amino acid variants ofsequence optimized anti-human PD-1 Nanobody monovalent module F023700706bound to human PD-1 expressed by the T-cell line, blocking PD-1'sinteraction with PD-L1; thereby, allowing the T-cell to respond to agreater degree to the T-cell receptor agonist based on inhibiting thePD-L1-mediated suppression (FIG. 12(E)).

Example 9 SEB Activated Human PBMC Assay

Thawed human peripheral blood mononuclear cells (PBMCs) into completeRPMI with 10% human serum (RPMI 1640 Glutamax (Thermo Fisher Scientific,catalog 11875085), 1× Penicillin-Streptomycin (Thermo Fisher Scientific,catalog 15140148), 1× beta-mercaptoethanol (Thermo Fisher Scientific,catalog 21985023), 1× HEPES (Thermo Fisher Scientific, catalog15630080), 1× sodium pyruvate (Thermo Fisher Scientific, catalog11360070), 1× non-essential amino acids (Thermo Fisher Scientific,catalog 11140076), human serum (Sigma, catalog H4522-100 MLlot#SLBP2783V). Suspended to 2.5E6 cells/mL and aliquot 100 uL to96-well U-bottom plate at 2.5E5 cells/well. Nanobodies were seriallydiluted using a 3-fold dilution series to prepared 4× working stocks andcreate a 6-point dose-response curve. Added 50 uL of Nanobody solutionto wells and incubated 15-30 minutes at room temperature while preparingstreptococcus exotoxin B (SEB) solution. Stock SEB (Toxin TechnologyInc, catalog BT202) was prepared by reconstituting SEB at 1 mg/mL indistilled water and freezing at −80° C. A 2 uM working solution was madeby adding 2 ul stock per mL media and the 2 uM working solution wasserially diluted using a 10-fold dilution series to make a titrationcurve. Added 50 uL to appropriate wells to make a titration curve as aninternal standard to confirm assay was performing as expected and todetermine the maximum response that could be elicited from differentdonors. The 2 uM SEB working solution was diluted to 40 nM SEB and 50 uLadded to wells to stimulate PBMC (final concentration of SEB is 10 nM).Incubate for 72 hours at 38° C. and 5% CO₂. Transferred top 100 uLsupernatant into a fresh 96-well plate and dilute 1:2 for IL-2 analysisusing MSD V-plex human IL-2 kit (MesoScale Discovery, K151QQD-1).Remainder stored at −80° C. The data from these experiments are setforth in FIG. 13(A-R).

This Example demonstrated that the bispecific anti-human PD-1/LAG-3Nanobodies F023700931 and F023700924 gave rise to increased IL-2 levelsfollowing T-cell receptor agonist stimulation of peripheral bloodmononuclear cell (PBMC) obtained from multiple donors (A-R).

Example 10 Mixed Lymphocyte Reaction (MLR) Assay

Human peripheral blood mononuclear cells (PBMCs) were purified fromleukopacks and frozen down in liquid nitrogen freezer. Frozen humanPBMCs were thawed, diluted in complete RPMI (RPMI+10% human serum),centrifuged at 450×g for 5 minutes, and the cell pellet resuspended withcomplete RPMI (Gibco RPMI 1640 media (Thermofisher Scientific;11875-119); human serum (Sigma-Aldrich; H4522-100 mL). Monocytes wereenriched using Human Monocyte Enrichment kit (STEMCELL technologies;19059). Cells were transferred to 6-well plates at 1×10⁶ cells/ml (5ml/well) in complete RPMI containing 100 ng/ml GM-CSF (R&D Systems;215-GM-110) and 50 ng/ml of human IL-4 (R&D Systems; 204-IL-010/CF).Monocytes were incubated at 37° C. for 5 days to allow for dendriticcell (DC) differentiation. Monocyte-derived dendritic cells (Mo-DC) wereharvested on Day 6, counted, and used in MLR assay as stimulators.

On the day of experiment initiation, frozen human PBMCs were thawed anddiluted two times in complete RPMI containing penicillin/streptomycin.CD4 T cells from each donor were enriched using EasySep Human CD4 T cellisolation kit (STEMCELL technologies; 17952). Isolated CD4 T cells weresuspended at 1×10⁶ cells/ml in complete RPMI. Mo-DC were mixed at 1:10ratio (1×10⁵ cells/ml) with CD4⁺T-cells (1x10⁶ cells/ml) and cellmixture plated in a U-bottom 96-well plate at 200 ul/well. Nanobodieswere serially diluted using a 4-fold dilution series and 5× workingstocks were prepared. 50 μL of each dilution was added to the 200 μLcultures to give 1× final concentration of nano/anti bodies. Culturesupernatants were collected at Day 5 post-experiment initiation for IFNγquantitation using V-plex Human Pro-inflammatory Panel I (MesoscaleDiscovery; K15052D-1). The data from these experiments are set forth inFIG. 14(A-F).

This Example demonstrated that the bispecific anti-human PD-1/LAG-3Nanobodies F023700931 and F023700924 gave rise to increased IFN-gammalevels following primary CD4 T-cell stimulation with allogeneic Mo-DCsobtained from different donors (a-f).

Example 11 Engineered 3A9.hLAG-3 Assay

3A9.hLAG3 is a human LAG-3 transgene expressing mouse 3A9 T-cellhybridoma. LK35.2 (ATCC; HB-98) is a mouse B-cell hybridoma that bearssurface I-Ad^(k) and I-Ed^(k) molecules that present a specific antigen(i.e. hen egg lysozyme (HEL) peptide DGSTDYGILQINSRWW) to the class IIrestricted 3A9 T-cell hybridoma. Counted 3A9.hLAG3 cells and resuspendat 4×10⁶ cells/ml of fresh medium [RPMI (Invitrogen, 61870-036)+10%FBS+Pen Strep]. Counted LK35.2 cells and resuspend at 1×10⁶ cells/ml offresh media. Added 25 μL 3A9.hLAG3 cells (1E5 cells) to wells in a96-well flat bottom tissue culture plate (Corning 3610). Prepared a 4×(200 uM) solution of human albumin (Sigma, A8763) and added 25 μL to thecells. Diluted Nanobodies to 5× concentration in complete medium and add20 ul of 5× Nanobody to cells. Incubated 3A9.hLAG3 cells with Nanobodiesfor 30 minutes at 37° C.

Prepared 500 μM HEL peptide (GenScript custom peptide) stock solutionand stored at −20 C. Diluted HEL peptide stock 1:20 (25 uM) in cellculture medium and then further diluted HEL peptide 1:833 onto LK35.2cells (final HEL concentration=−30 nM). Incubated LK35.2 cells withpeptide for 30 minutes at 37C. Added 33 μL peptide treated LK35.2 cellsto the 96-well plate containing Nanobody treated 3A9.hLAG-3 cells.Incubated culture at 37° C. for 24 hours. Spinned plates at 300×g for 5minutes and collected supernatant for IL-2 analysis (IL-2 MesoscaleV-Plex; Mesoscale K152QQD-4). The data from these experiments were setforth in FIG. 15(A-D).

This Example demonstrated that the anti-human LAG-3 Nanobody monovalentmodule F023700842 bound to human LAG-3 expressed by the T-cell line,blocked LAG-3's interaction with MHC Class II expressed by theco-culture cell line to relieve the suppression that MHC Class II wasproviding to the T-cell; thereby, allowing the T-cell to respond to agreater degree to the T-cell receptor agonist based on inhibiting theMHC Class II-mediated suppression. The potency of inhibiting the MHCClass II by the anti-human LAG-3 Nanobody monovalent module F023700842was similiar to that of the parental Nanobody F023700656 from which itwas derived (FIG. 15(A)). Furthermore, this Example demonstrated thatthe bispecific anti-human PD-1/LAG-3 Nanobodies F023700931 andF023700924 bound to human LAG-3 expressed by the T-cell line, blockedLAG-3′s interaction with MHC Class II; thereby, allowing the T-cell torespond to a greater degree to the T-cell receptor agonist based oninhibiting the MHC Class II-mediated suppression (FIG. 15(B)). Themonospecific, bivalent controls anti-human PD-1 F023700933 andanti-human LAG-3 F023700962 were also shown. Additionally, this Exampleshowed that the bispecific anti-human PD-1/LAG-3 Nanobody F023700969 andF023700970 containing 20GS or 9GS linkers, respectively, had similiarpotency as the parent F023700924 molecule containing 35GS linkers (FIG.15(C)). Similarly, the bispecific anti-human PD-1/LAG-3 NanobodyF023701016 and F023701017, containing 20GS or 9GS linkers, respectively,had similiar potency to the parent F023700931 molecule containing 35GSlinkers (FIG. 15(D)).

Example 12 Engineered Bispecific Jurkat.hPD-1.LAG-3 Bioassay

Generation of Jurkat.LAG-3.PD-1 cells (DT1088-Clone G10PD1)

A human LAG-3 transgene was introduced into Jurkat cells (Je6.2.11)using a retroviral delivery system with puromycin as a selection marker.Limiting dilution was conducted to pick a clone (DT1088G10) with optimalLAG-3 and CD3 expression. A human PD-1 transgene was introduced intoDT1088G10 clone using a Lentiviral delivery system with Genticin as aselection marker. Cells were FACS-sorted for high LAG-3 and PD-1expression. Sorted cell pool was maintained and subcultured in RPMIcomplete media [RPMI (Corning 10-040-CV) supplemented with 10% FBS(Hyclone SH30910.03)+1 mM sodium pyruvate (BioWhittaker, 13-115E)+2 mML-glutamine (Corning CellGro, 25-005-CI) +10 mM HEPES (Corning,26-060-CI)+1×non-essential amino acids (Sigma, M7145)+0.2 ug/mlpuromycin (Sigma P9620), +0.5 mg/ml G418 (Gibco 10131-027)].

Generation of the Raji.PD-L1 Expressing Cells

A human PD-L1 transgene was introduced into Raji cells (ATCC CCL-86)using a retroviral delivery system with puromycin as a selection marker.The cells were sorted to enrich for PD-L1 and Class-II (endogenous)expression. The enriched pool of cells were maintained and subculturedin RPMI complete media (RPMI media supplemented with 10% Hyclone FBS+2mM L-glutamine+10 mM HEPES+0.25 ug/ml puromycin).

Stock SED toxin was prepared by reconstituting SED (Toxin technologyDT303) at 1 mg/ml in sterile distilled water and storing at −80° C.Raji.PD-L1 cells were preloaded with toxin by incubating a suspension of0.87×10⁶ Raji-PD-L1 cells/ml with SED toxin (1.3×=130 ng/ml, final 100ng/ml) in RPMI media containing 10% dialyzed FBS (Hyclone SH30079.03)for 30 minutes in a 37° C. incubator. A suspension of 8X10⁶Jurkat.LAG-3.PD-1 cells/ml in RPMI media containing 10% dialyzed FBS wasprepared. Nanobodies were prepared as an 8 point, 4-fold dose titration(12×=120 μg/ml) of Nanobodies with a starting assay concentration of 10ug/ml.

45 ul Jurkat.LAG-3.PD-1 cell suspension (8×10⁶ cells/ml) was incubatedwith 45 ul of Nanobody titration at room temperature for 30 minutes in around bottom plate (Corning 3359). At the end of 30 minutes, 36 uL humanalbumin (Sigma, A8763; 15×=450 uM to get a final assay concentration of30 uM) was added to the Jurkat.LAG-3.PD-1+Nanobody mixture. 115 ulSED-loaded Raji.PD-L1 cells were added to assay plates (Thermoscientific#167008). To the SED-loaded Raji.PD-L1 cells, 35 uL mixture ofJurkat.LAG-3.PD-1+Nanobodies+albumin was carefully layered. Assay plateswere incubated for 24 hours at 37° C. in a 5% CO₂ incubator. Following24 hour incubation, 75 uL supernatant was transferred to a plate(Corning#3605) and frozen at −80 degrees. Samples were analyzed usingthe MSD IL-2 V-plex kit (Mesoscale devices Cat#K151QQD). EC50 valueswere calculated using the GraphPad prism Software. The data from theseexperiments are set forth in FIG. 16(A-B).

This Example demonstrated that the bispecific anti-human PD-1/LAG-3Nanobodies F023700931 and F023700924 bound to both human PD-1 and humanLAG-3 expressed by the T-cell line, blocked PD-1's interaction withPD-L1, blocks LAG-3's interaction with MHC Class II; thereby, allowingthe T-cell to respond, to a greater degree, to the T-cell receptoragonist based on inhibiting the dual PD-L1-mediated and MHC ClassII-mediated suppression (FIG. 16(A)). The monospecific, bivalentcontrols anti-human PD-1 F023700933 and anti-human LAG-3 F023700862 wereonly able to block one of the two inhibitory mechanisms individuallyleading to the T-cell responding in a much more modest fashion to theT-cell receptor agonist. Additionally, this Example showed that thebispecific anti-human PD-1/LAG-3 Nanobody F023700969 and F023700970containing 20GS or 9GS linkers, respectively, had similiar potency tothe parent F023700924 molecule containing 35GS linkers. Similarly, thebispecific anti-human PD-1/LAG-3 Nanobody F023701016 and F023701017containing 20GS or 9GS linkers, respectively, had similiar potency asthe parent F023700931 molecule containing 35GS linkers (FIG. 16(B)).

Example 13 Human T-cell Clone+JY.hPD-L1 Assay

Generation and Culture of Human CD4+ T Cell Clone

MHC class II allo-antigen specific CD4+ T cell clone BC4-49 wasgenerated by 2 rounds of mixed leukocyte reaction with theEBV-transformed B-cell line JY and cloned by limiting dilution. Theclone was re-stimulated with allo-specific antigens at an interval ofevery 2 weeks and cultured in Yssel's medium (IMDM, Gibco 12440-053;human serum AB, Gemimi 100512; penicilin/streptomycin, Mediatech30-002-CI; human albumin, Sigma A9080; ITS-X, Gibco 51500056;Transferin, Roche 10652202001; PA Bioxtra Sigma p5585; LA-OA-Albumin,Sigma L9655). Fresh PBMCs were isolated from two human buffy coatsprovided by Stanford Blood Center and pooled at 1:1 cell ratio. PBMCswere irradiated in a gamma irradiator at dose 4000 rads before use.Wildtype JY cells were prepared and irradiated at dose 5000 rads. T cellclones were cultured with feeders in 24-well plate at lmL per well withfinal concentrations of CD4+ T cells 0.2×10⁶ /mL, irradiated PBMCs1×10⁶/mL, irradiated JY 0.1×10⁶/mL, and 100 ng/mL PHA (Sigma L9017).Recombinant human IL-2 (R&D Systems; 202-IL/CF) was added at finalconcentration of 100 ng/mL on day 3 after re-stimulation, and wasreplenished every 3-4 days throughout the expansion. Cells were passagedto an optimal concentration between 0.5-1.0×10⁶/mL. On day 7 afterre-stimulation, abundant level of LAG-3 and moderate level of PD-1 wereexpressed on T cell surface.

Human CD4+ T Cell Functional Assay

Allo-antigen specific CD4+ T cells were harvested from 24 well cultureplates on day 7 after antigen re-stimulation, then washed twice with 20mL PBS (Hyclone, SH3002802) containing 2 mM EDTA (Invitrogen, 15575-38)by centrifugation. The pellets were re-suspended into single cellsuspension in Yssel's medium. Tested Nanobodies were titrated by 5-foldserially dilutions in Yssel's medium starting from highest concentrationof 133 nM with total 7 dilutions in a volume of 100 μL in 96 wellU-bottom culture plates (Falcon, 353077). Fifty microliters of T cellsuspension at a density of 4×10⁵ cells/mL was added into wellscontaining titrated Nanobodies. The Nanobody/T cell mixture waspre-incubated for 1 hour in an incubator at 37° C. with 5% CO₂. HumanPD-L1 transgene expressing JY cells (JY.hPD-L1) were used in co-culturesto provide allo-specific antigens. JY.hPD-L1 cells cultured in T-75flask (Thermo Scientific, 156499) in RPMI medium (Corning Cellgro,10-040-CV) with 10% FCS were harvested and irradiated in a gammairradiator at a dose of 5000 rads, then washed twice with PBS containing2 mM EDTA by centrifugation. The pellet was re-suspended with Yssel'smedium, and filtered with 40 μm cell strainer before plating. 50 μL/wellof JY.hPD-L1 suspension at a concentration of 2×10⁵ cells/mL wasdispensed into pre-incubated Nanobody-T cells mixture, with T cell toJY.hPD-L1 cell ratio at 2:1. All conditions were run in duplicates.After approximately 3-day culture, 100 μL of supernatant per well washarvested for human IFNγ quantification. Human IFNγ ELISA was performedto assess IFNγ level on pooled supernatant from duplicates by usinghIFNγ Quantikine kit (R&D Systems, SIF50). Assays were run following thestandard protocol provided by manufacturer. EC50 values were calculatedusing the GraphPad prism software. The data from these experiments areset forth in FIG. 17(A-C).

This Example demonstrated that the bispecific anti human PD 1/LAG-3Nanobodies F023700931 and F023700924 bound to both human PD-1 and humanLAG 3 expressed by the T-cell clone, blocked PD-1's interaction withPD-L1, blocked LAG-3's interaction with MHC Class II; thereby, allowingthe T-cell to respond, to a greater degree, to the allogeneicstimulation based on inhibiting the dual PD-L1-mediated and MHC ClassII-mediated suppression (FIG. 17(A)). The monospecific, bivalentcontrols anti-human PD-1 F023700933 and anti-human LAG-3 F023700862 wereonly able to block one of the two inhibitory mechanisms individuallyleading to the T-cell responding, in a much more modest fashion, to theallogeneic stimulation. Additionally, this Example showed that thebispecific anti-human PD-1/LAG-3 Nanobody F023700969 and F023700970,containing 20GS or 9GS linkers, respectively, had similiar potency asthe parent F023700924 molecule containing 35GS linkers (FIG. 17(C)).Similarly, the bispecific anti human PD-1/LAG-3 Nanobody F023701016 andF023701017, containing 20GS or 9GS linkers, respectively, had similiarpotency as the parent F023700931 molecule containing 35GS linkers (FIG.17(B)).

Example 14 Evaluation of Pre-Antibody Binding to F023700924 andF023700931

Binding of pre-existing antibodies to Nanobodies captured on human serumalbumin (HSA) was evaluated using the ProteOn XPR36 (Bio-RadLaboratories, Inc.). PBS/Tween (phosphate buffered saline, pH 7.4,0.005% Tween 20) was used as running buffer and the experiments wereperformed at 25° C. The ligand lanes of a ProteOn GLC Sensor Chip wereactivated with EDC/NHS (flow rate 30 μL/min) and human serum albumin(has) was injected at 10 μg/ml in ProteOn Acetate buffer pH 4.5 (flowrate 100 μl/min) to render immobilization levels of approximately 3600RU. After immobilization, surfaces were deactivated with ethanolamineHCl (flow rate 30 μL/min). Nanobodies were injected for 2 minutes at 45μL/min over the HSA surface to render a Nanobody capture level ofapproximately 600 RU for trivalent F023700924 and approximately 1000RUfor pentavalent F023700931. The samples containing pre-existingantibodies were diluted 1:10 in PBS-Tween20 (0.005%) before beinginjected for 2 minutes at 45 μl/min followed by a subsequent 400 secondsdissociation step. After each cycle (i.e. before a new Nanobody captureand blood sample injection step), the HSA surfaces were regenerated witha 2 minute injection of HCl (100 mM) at 45 μl/min. Sensorgram processingand data analysis was performed with ProteOn Manager 3.1.0 (Bio-RadLaboratories, Inc.). Sensorgrams showing pre-existing antibody bindingwere obtained after double referencing by subtracting 1) Nanobody-HSAdissociation and 2) non-specific binding to reference ligand lanecontaining HSA only. Binding levels of pre-existing antibodies weredetermined by setting report points at 125 seconds (5 seconds after endof association). As a reference, the samples containing pre-existingantibodies were also tested for binding to a trivalent Nanobody notmodified for reducing the binding of these pre-existing antibodies(T013700112). The data from these experiments are set forth in FIG.19(A-I).

Bispecific anti-human PD-1/LAG-3 Nanobodies F023700924 had threeNanobody modules (one PD-1 module, one LAG-3 module, and oneanti-albumin module); whereas, F023700931 has five Nanobody modules(duplicate PD-1 modules, duplicate LAG-3 modules, and one anti-albuminmodule). One component of the sequence optimization approach was toincorporate amino acid substitutions into the Nanobody framework thatdecreased the reactivity present in human sera to the Nanobody modules.This Example demonstrated the pre-existing reactivity found in a panelof serum from healthy human donors to the 3-module bispecific anti-humanPD-1/LAG-3 Nanobody F023700924 was reduced compared to the pre-existingreactivity to a different 3-module Nanobody T013700112 construct thatdoes not contain these framework amino acid substitutions at twotimepoints. Reactivity of the 5-module bispecific anti-human PD-1/LAG-3Nanobodies F023700931 was higher than the 3-module F023700924 (FIG.19(A-B)). In addition, this Example demonstrated the pre-existingreactivity found in a panel of serum from cancer patients to the3-module bispecific anti-human PD-1/LAG-3 Nanobodies F023700924 isreduced compared to the pre-existing reactivity to a different 3-moduleNanobody T013700112 construct that does not contain these frameworkamino acid substitutions at two timepoints. Reactivity of the 5-modulebispecific anti-human PD-1/LAG-3 Nanobodies F023700931 was higher thanthe 3-module F023700924 (FIG. 19(C-D)). Lastly, the reactivity of thepanel of serum from cancer patients towards F023700924 and F023700931was separated based on cancer indication (FIG. 19(E-I)).

Example 15 Affinity Measurement of Sequence Optimized Human LAG-3Nanobodies by Surface Plasmon Resonance

Kinetic analysis of Nanobodies was performed by surface plasmonresonance (SPR) technology, using the Biacore T100 instrument (GEHealthcare). HBS-EP+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05%v/v Surfactant P20) was used as running buffer and the experiments wereperformed at 25° C. Two flow channels of a Series S Sensor Chip CM5 wereactivated with EDC (200 mM) /NHS (50 mM) and anti-Human IgG(Fc) (GEHealthcare, BR100839) was injected at 5 μg/ml in the suppliedimmobilization buffer (10 mM sodium acetate pH5.0) to renderimmobilization levels of approximately 2000 RU. After immobilization,surfaces were deactivated with 1M ethanolamine/HCl (pH 8.5). The flowrate during the sensor preparation was set on 5 μL/min.

In the kinetic analysis, 62.5 nM human LAG3-hFc was injected for 1minute at 10 μL/min over one anti-Human IgG(Fc) surface to render acapture level of approximately 600 RU. 2.5 fold serial dilutions of theNanobodies (i.e., 5 μM down to 3.3 nM for F023700842) were made inrunning buffer before being injected for 2 minutes at 45 μL/min followedby a subsequent 900 seconds dissociation. After each cycle (i.e. beforea new human LAG3-hFc capture and Nanobody injection step) theanti-hIgG(Fc) surfaces were regenerated with a 2 minute injection ofMgCl₂ (3M) at 10 μL/min.

Sensorgram processing and data analysis was performed with Biacore T100Evaluation Software Version 2.0.4 (GE Healthcare). Sensorgrams showingNanobody binding were obtained after double referencing bysubtracting 1) human LAG3-hFc/anti-hlgG(Fc) dissociation and 2)non-specific binding to reference flow channel. Processed curves wereevaluated via fitting with the model ‘Langmuir with Mass Transport’.

This Example demonstrated that the anti-human LAG-3 monovalent NanobodyF023700842 bound with high affinity to human LAG-3.

TABLE E Affinity Measurement of Human LAG-3 Nanobodies by SurfacePlasmon Resonance. ka (1/Ms) kd (1/s) KD (M) F023700929 Human PD-1-Fc2.0E+06 5.9E−04 2.9E−10 F023701192 2.5E+06 3.2E−03 1.3E−09 F0237011933.2E+06 7.5E−03 2.4E−09 F023700842 Human LAG-3-Fc 9.2E+05 1.9E−022.1E−08 F023700924 Human Serum 6.4E+04 7.7E−03 1.2E−07 F023700931Albumin 3.2E+04 8.2E−03 2.5E−07 F023700924 Rhesus Serum 6.9E+04 7.9E−031.2E−07 F023700931 Albumin 3.2E+04 8.4E−03 2.7E−07

Example 16 Affinity Measurement of Sequence Optimized Human PD-1Nanobodies by Surface Plasmon Resonance

Kinetic analysis of the Nanobodies was performed by SPR technology,using the Biacore T100 instrument (GE Healthcare). HBS-EP+ (0.01 M HEPESpH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20) was used asrunning buffer and the experiments were performed at 25° C. Two flowchannels of a Series S Sensor Chip CMS were activated with EDC (200mM)/NHS (50 mM). hPD-1-hFc was injected (4 times) at 5 μg/ml in suitableimmobilization buffer (10 mM sodium acetate pH4.5) over one surface torender immobilization levels of 303 RU. After immobilization, surfaceswere deactivated with 1M ethanolamine/HC1 (pH 8.5). The flow rate duringthe sensor preparation was set on 5 μl/min.

In the kinetic analysis, 2.5 Fold serial dilutions of the Nanobodies(i.e. 1 μM down to 0.46 nM for F023700929, F023701192 and F023701193)were made in running buffer before being injected for 2 minutes at 45μl/min followed by subsequent 900 seconds dissociation. After each cycle(i.e. before a new Nanobody injection step) the hPD1-hFc surfaces wereregenerated with a 1 minute injection 10 mM Glycine pH1.5 at 45 μl/min.Sensorgram processing and data analysis was performed with Biacore T100Evaluation Software Version 2.0.4 (GE Healthcare). Sensorgrams showingNanobody binding were obtained after double referencing bysubtracting 1) non-specific binding to reference flow channel and 2)HBS-EP+injection. Processed curves were evaluated via fitting with themodel ‘Langmuir with Mass Transport’.

This Example demonstrated that the anti-human PD-1 monovalent NanobodyF023700929 bound with high affinity to human PD-1. In addition, thisExample demonstrated that additional amino acid variants of sequenceoptimized anti-human PD-1 monovalent F023700929 (i.e., F023701192 andF023701193) bound with high affinity to human PD-1.

TABLE F Affinity Measurement of Sequence Optimized Human PD-1 Nanobodiesby Surface Plasmon Resonance. ka T kd T Rmax T KD kt Chi² Chi²/Rmax(1/Ms) (ka) (1/s) (kd) (RU) (Rmax) (M) (RU/Ms) (RU²) (%) F0237009292.0E+06 220 5.9E−04 150 32.0 500 2.9E−10 2.9E+15 1.8 5 F0237011922.5E+06 63 3.2E−03 66 27.9 380 1.3E−09 8.7E+07 0.8 3 F023701193 3.2E+0649 7.5E−03 49 29.5 450 2.4E−09 4.2E+07 1.2 4

Example 17 Affinity Measurement of Sequence Optimized MultispecificHuman PD-1/LAG-3 to Albumin of Different Species by Surface PlasmonResonance

Kinetic analysis of the Nanobodies was performed by SPR technology,using the Biacore T100 instrument (GE Healthcare). HBS-EP+ (0.01M HEPESpH 7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20) was used asrunning buffer and the experiments were performed at 25° C. Four flowchannels of a Series S Sensor Chip CMS were activated with EDC (200mM)/NHS (50 mM) and Human Serum Albumin (Sigma, cat. A3782, lot.SLBD7204V) or Rhesus Serum Albumin (BioWorld, cat. 22070099-1,lot.L15091001DA) were injected at 5 μg/ml in 10 mM Sodium Acetate buffer pH4.5 to render immobilization levels between 179 and 312 RU on three flowchannels, respectively. After immobilization, surfaces were deactivatedwith 1M ethanolamine/HCl (pH 8.5). The flow rate during the sensorpreparation was set on 5 μl/min.

In the kinetic analysis, three fold serial dilutions of the Nanobodies(i.e. 6 μM down to 2.7 nM) were prepared in running buffer before beinginjected for 2 minutes at 45 μl/min followed by subsequent 900 secondsdissociation. After each cycle (i.e. before a new Nanobody concentrationwas injected) all surfaces were regenerated with a 10 second injectionof 10 mM Glycine pH 1.5 at 100 μl/min. Sensorgram processing and dataanalysis was performed with Biacore T100 Evaluation Software Version2.0.4 (GE Healthcare). Sensorgrams showing Nanobody binding wereobtained after double referencing by subtracting 1) non-specific bindingto reference flow channel and 2) average of two HBS-EP+ injections onthe specific flow channel. Processed curves were evaluated via fittingwith the model ‘Langmuir with Mass Transport’.

This Example demonstrated that the bispecific anti human PD 1/LAG-3Nanobodies F023700931 and F023700924 bound to both human albumin andrhesus albumin with similar affinity.

TABLE G Affinity Measurement of Sequence Optimized Multispecific HumanPD- 1/LAG-3 to Albumin of Different Species by Surface PlasmonResonance. HSA RhSA MSA ka kd KD ka kd KD ka kd KD Nanobody Valency(1/Ms) (1/s) (M) (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) F023700924-CHO-EXPITrimer 6.4E+04 7.7E−03 1.2E−07 6.9E+04 7.9E−03 1.2E−07 >6.0E+06 >6.0E−01−>1.9E−06 F023700931-CHO-EXPI Pemamer 3.2E+04 8.2E−03 2.5E−07 3.2E+048.4E−03 2.7E−07 >6.0E+06 >6.0E−01 −>1.1E−06

We claim:
 1. A multispecific binder comprising: (a) an immunoglobulinsingle variable domain (ISVD) that binds to programed cell death protein1 (PD1) comprising a CDR1 comprising the amino acid sequence set forthin SEQ ID NO: 3 or SEQ ID NO: 6; a CDR2 comprising the amino acidsequence set forth in SEQ ID NO: 4 or SEQ ID NO: 7; and a CDR3comprising the amino acid sequence set forth in SEQ ID NO: 5; and (b) anISVD that binds to lymphocyte activation gene 3 (LAG3) comprising a CDR1comprising the amino acid sequence set forth in SEQ ID NO: 65 or SEQ IDNO: 154; a CDR2 comprising the amino acid sequence set forth in SEQ IDNO: 66 or SEQ ID NO: 139; and a CDR3 comprising the amino acid sequenceset forth in SEQ ID NO:
 67. 2. The multispecific binder of claim 1,wherein the ISVD that binds to PD1 comprises: the amino acid sequenceset forth in SEQ ID NO: 1 or 2 wherein the sequence comprises a mutationat residue 11 and/or 89; wherein said residue numbers are Kabat residuenumbers.
 3. The multispecific binder of claim 1, wherein the ISVD thatbinds PD1 comprises the amino acid sequenceDVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQAPGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVYLQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTLVTVSS

(SEQ ID NO: 57, or said sequence comprising a D1E mutation);EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDK HQSSWYDYWGQGTLVTVSS

(SEQ ID NO: 98, or said sequence comprising a E1D mutation);EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK HQSSWYDYWGQGTLVTVSS

(SEQ ID NO: 99, or said sequence comprising a E1D mutation);DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS

(SEQ ID NO: 103, or said sequence comprising a D1E and/or N73Smutation); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS

(SEQ ID NO: 104, or said sequence comprising a D1E mutation);DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS

(SEQ ID NO: 105, or said sequence comprising a D1E mutation),DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS

 (amino acids 1-119 of SEQ ID NO: 113 , or said sequence comprising a MEmutation); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS

 (amino acids 1-119 of SEQ ID NO: 117, or said sequence comprising a D1Emutation); DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDSSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS

 (amino acids 1-119 of SEQ ID NO: 121, or said sequence comprising a D1Emutation).
 4. The multispecific binder of claim 1, wherein the ISVD thatbinds LAG3 comprises the amino acid sequence set forth in SEQ ID NO: 64.5. The multispecific binder of claim 1, wherein the multispecific binderfurther includes an ISVD that binds to human serum albumin comprisingCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60 orSEQ ID NO: 151; CDR2 comprising the amino acid sequence set forth in SEQID NO: 61 or SEQ ID NO: 152; and CDR3 comprising the amino acid sequenceset forth in SEQ ID NO:
 62. 6. A multispecific binder comprising: (a) animmunoglobulin single variable domain (ISVD) that binds to programmedcell death protein 1 (PD1) comprising: a CDR1 comprising the amino acidsequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); a CDR2comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4) orVITXSGGITY (SEQ ID NO: 7); and a CDR3 comprising the amino acid sequenceDKHQSSXYDY (SEQ ID NO: 5); (b) an immunoglobulin single variable domainthat binds to lymphocyte activation gene 3 (LAG3) comprising: a CDR1comprising the amino acid sequence GRTFSDYVMG (SEQ ID NO: 139) or DYVMG(amino acids 6-10 of SEQ ID NO: 65); a CDR2 comprising the amino acidsequence AISESGGRTHYADSVKG (SEQ ID NO: 66) or AISESGGRTH (SEQ ID NO:66); and a CDR3 comprising the amino acid sequence TLLWWTSEYAPIKANDYDY(SEQ ID NO: 67); and, (c) a half-life extender and a C-terminalextender.
 7. The multispecific binder of claim 6, wherein (a) theimmunoglobulin single variable domain that binds to PD1 comprises theamino acid sequence (SEQ ID NO: 57)DVQLVESGGG VVQPGGSLRL SCAASGSIAS IHAMGWFRQAPGKEREFVAV ITWSGGITYY ADSVKGRFTI SRDNSKNTVYLQMNSLRPED TALYYCAGDK HQSSWYDYWG QGTLVTVSS; (SEQ ID NO: 98)EVQLVESGGGLVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAIYYCAGDK HQSSWYDYWGQGTLVTVSS;(SEQ ID NO: 99) EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWERQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK HQSSWYDYWGQGTLVTVSS;

(amino acids 1-119 of SEQ ID NO: 101)DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK HQSSWYDYWGQGTLVTVSS;

(amino acids 1-119 of SEQ ID NO: 101); (SEQ ID NO: 103)DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS;(SEQ ID NO: 104) DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS;or (SEQ ID NO: 105) DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDK HQSSFYDYWGQGTLVTVSS;

 and, (b) the immunoglobulin single variable domain that binds to LAG3comprises the amino acid sequence (SEQ ID NO: 64)EVQLVE SGGGVVQPGG SLRLSCAASG RTFSDYVMGW FRQAPGKEREFVAAISESGG RTHYADSVKG RFTISRDNSK NTLYLQMNSLRPEDTALYYC ATTLLWWTSE YAPIKANDYD YWGQGTLVTV SS;

10 orDVQLVESGGGVVQPGGSLRLSCAASGRTFSDYVMGWFRQAPGKEREFVAAISESGGRTHYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCATTLLWWTSEYAPIKANDYDYWGQGTLVTVSS (amino acids 1-128 of SEQ IDNO: 96).
 8. The multispecific binder of claim 6, wherein the C-terminalextender is an alanine.
 9. The multispecific binder of claim 6, whereinthe multispecific binder comprises: a PD1 ISVD comprising the amino acidsequence set forth in SEQ ID NO: 98; a 35 GS linker comprising the aminoacid sequence set forth in SEQ ID NO: 58; a PD1 ISVD comprising theamino acid sequence set forth in SEQ ID NO: 98; a 35 GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3ISVD comprising the amino acid sequence set forth in SEQ ID NO: 63; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58;a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO:63; a 35 GS linker comprising the amino acid sequence set forth in SEQID NO: 58; a HSA ISVD comprising the amino acid sequence set forth inSEQ ID NO: 142; and a C-terminal Alanine; or, a PD1 ISVD comprising theamino acid sequence set forth in SEQ ID NO: 57; a 35 GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 58; a PD1ISVD comprising the amino acid sequence set forth in SEQ ID NO: 99; a 35GS linker comprising the amino acid sequence set forth in SEQ ID NO: 58;a LAG3 ISVD comprising the amino acid sequence set forth in SEQ ID NO:64; a 35 GS linker comprising the amino acid sequence set forth in SEQID NO: 58; a LAG3 ISVD comprising the amino acid sequence set forth inSEQ ID NO: 64; a 35 GS linker comprising the amino acid sequence setforth in SEQ ID NO: 58; a HSA ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 142; and a C-terminal Alanine; or, a PD1 ISVDcomprising the amino acid sequence set forth in SEQ ID NO: 57; a 20 GSlinker comprising the amino acid sequence set forth in SEQ ID NO: 100; aPD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO: 99;a 20 GS linker comprising the amino acid sequence set forth in SEQ IDNO: 100; a LAG3 ISVD comprising the amino acid sequence set forth in SEQID NO: 64; a 20 GS linker comprising the amino acid sequence set forthin SEQ ID NO: 100; a LAG3 ISVD comprising the amino acid sequence setforth in SEQ ID NO: 64; a 20 GS linker comprising the amino acidsequence set forth in SEQ ID NO: 100; a HSA ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 142; and a C-terminal Alanine; or,a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO:57; a 9 GS linker comprising the amino acid sequence set forth in SEQ IDNO: 125; a PD1 ISVD comprising the amino acid sequence set forth in SEQID NO: 99; a 9 GS linker comprising the amino acid sequence set forth inSEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forthin SEQ ID NO: 64; a 9 GS linker comprising the amino acid sequence setforth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 64; a 9 GS linker comprising the amino acidsequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 142; and a C-terminal Alanine; or,a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO:57; a 35GS linker comprising the amino acid sequence set forth in SEQ IDNO: 58; a LAG3 ISVD comprising the amino acid sequence set forth in SEQID NO: 64; a 35GS linker comprising the amino acid sequence set forth inSEQ ID NO: 58; a HSA ISVD comprising the amino acid sequence set forthin SEQ ID NO: 142; and a C-terminal Alanine; or, a PD1 ISVD comprisingthe amino acid sequence set forth in SEQ ID NO: 57; a 20GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 100; a LAG3ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a20GS linker comprising the amino acid sequence set forth in SEQ ID NO:100; a HSA ISVD comprising the amino acid sequence set forth in SEQ IDNO: 142; and a C-terminal Alanine; or, a PD1 ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 57; a 9GS linker comprising theamino acid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprisingthe amino acid sequence set forth in SEQ ID NO: 64; a 9GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 125; a HSAISVD comprising the amino acid sequence set forth in SEQ ID NO: 142; anda C-terminal Alanine; or, a PD1 ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 105; a 35GS linker comprising the amino acidsequence set forth in SEQ ID NO: 58; a PD1 ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 105 (wherein the Asp at position 1is substituted with a Glu); a 35GS linker comprising the amino acidsequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising theamino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprisingthe amino acid sequence set forth in SEQ ID NO: 142; and a C-terminalAlanine; or, a PD1 ISVD comprising the amino acid sequence set forth inSEQ ID NO: 105; a 9GS linker comprising the amino acid sequence setforth in SEQ ID NO: 125; a PD1 ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 105 (wherein the Asp at position 1 issubstituted with a Glu); a 9GS linker comprising the amino acid sequenceset forth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acidsequence set forth in SEQ ID NO: 64; a 9GS linker comprising the aminoacid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprising theamino acid sequence set forth in SEQ ID NO: 142; and a C-terminalAlanine; or, a PD1 ISVD comprising the amino acid sequence set forth inSEQ ID NO: 105; a 35GS linker comprising the amino acid sequence setforth in SEQ ID NO: 58; a LAG3 ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 64; a 35GS linker comprising the amino acidsequence set forth in SEQ ID NO: 58; a HSA ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 142; and a C-terminal Alanine; or,a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO:105; a 9GS linker comprising the amino acid sequence set forth in SEQ IDNO: 125; a LAG3 ISVD comprising the amino acid sequence set forth in SEQID NO: 64; a 9GS linker comprising the amino acid sequence set forth inSEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence set forthin SEQ ID NO: 142; and a C-terminal Alanine; or, a PD1 ISVD comprisingthe amino acid sequence set forth in SEQ ID NO: 104; a 35GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 58; a PD1ISVD comprising the amino acid sequence set forth in SEQ ID NO: 104(wherein the Asp at position 1 is substituted with a Glu); a 35GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a35GS linker comprising the amino acid sequence set forth in SEQ ID NO:58; a HSA ISVD comprising the amino acid sequence set forth in SEQ IDNO: 142; and a C-terminal Alanine; or, a PD1 ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 104; a 9GS linker comprising theamino acid sequence set forth in SEQ ID NO: 125; a PD1 ISVD comprisingthe amino acid sequence set forth in SEQ ID NO: 104 (wherein the Asp atposition 1 is substituted with a Glu); a 9GS linker comprising the aminoacid sequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising theamino acid sequence set forth in SEQ ID NO: 64; a 9GS linker comprisingthe amino acid sequence set forth in SEQ ID NO: 125; a HSA ISVDcomprising the amino acid sequence set forth in SEQ ID NO: 142; and aC-terminal Alanine; or, a PD1 ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 104; a 35GS linker comprising the amino acidsequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 64; a 35GS linker comprising theamino acid sequence set forth in SEQ ID NO: 58; a HSA ISVD comprisingthe amino acid sequence set forth in SEQ ID NO: 142; and a C-terminalAlanine; or, a PD1 ISVD comprising the amino acid sequence set forth inSEQ ID NO: 104; a 9GS linker comprising the amino acid sequence setforth in SEQ ID NO: 125; a LAG3 ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 64; a 9GS linker comprising the amino acidsequence set forth in SEQ ID NO: 125; a HSA ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 142; and a C-terminal Alanine; or,a PD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO:103 (wherein the Asn at position 73 is substituted with Ser); a 35GSlinker comprising the amino acid sequence set forth in SEQ ID NO: 58; aPD1 ISVD comprising the amino acid sequence set forth in SEQ ID NO:103(wherein the Asp at position 1 is substituted with Glu and the Asn atposition 73 is replaced with Ser); a 35GS linker comprising the aminoacid sequence set forth in SEQ ID NO: 58; a LAG3 ISVD comprising theamino acid sequence set forth in SEQ ID NO: 64; a 35GS linker comprisingthe amino acid sequence set forth in SEQ ID NO: 58; a HSA ISVDcomprising the amino acid sequence set forth in SEQ ID NO: 142; and aC-terminal Alanine; or, a PD1 ISVD comprising the amino acid sequenceset forth in SEQ ID NO: 103 (wherein the Asn at position 73 issubstituted with Ser); a 9GS linker comprising the amino acid sequenceset forth in SEQ ID NO: 125; a PD1 ISVD comprising the amino acidsequence set forth in SEQ ID NO:103 (wherein the Asp at position 1 issubstituted with Glu and the Asn at position 73 is substituted withSer); a 9GS linker comprising the amino acid sequence set forth in SEQID NO: 125; a LAG3 ISVD comprising the amino acid sequence set forth inSEQ ID NO: 64; a 9GS linker comprising the amino acid sequence set forthin SEQ ID NO: 125; a HSA ISVD comprising the amino acid sequence setforth in SEQ ID NO: 142; and a C-terminal Alanine, or, a PD1 ISVDcomprising the amino acid sequence set forth in SEQ ID NO: 103 (whereinthe Asn at position 73 is substituted with Ser); a 35 GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 58; a LAG3ISVD comprising the amino acid sequence set forth in SEQ ID NO: 64; a35GS linker comprising the amino acid sequence set forth in SEQ ID NO:58; a HSA ISVD comprising the amino acid sequence set forth in SEQ IDNO: 142; and a C-terminal Alanine; or, a PD1 ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 103 (wherein the Asn at position73 is substituted with Ser); a 9GS linker comprising the amino acidsequence set forth in SEQ ID NO: 125; a LAG3 ISVD comprising the aminoacid sequence set forth in SEQ ID NO: 64; a 9 GS linker comprising theamino acid sequence set forth in SEQ ID NO: 125; a HSA ISVD comprisingthe amino acid sequence set forth in SEQ ID NO: 142; and a C-terminalAlanine; and, wherein said residue numbers are Kabat residue numbers.10. The multispecific binder of claim 6, wherein the multispecificbinder comprises the amino acid sequence set forth in SEQ ID NO: 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, or
 124. 11. The multispecific binder of claim 6, whereinthe half-life extender is a human serum albumin ISVD that binds to humanserum albumin comprising CDR1 comprising the amino acid sequenceGFTFSSFGMS (SEQ ID NO: 60) or SFGMS (SEQ ID NO: 151); CDR2 comprisingthe amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 61) or SISGSGSDTL(SEQ ID NO: 152); and CDR3 comprising the amino acid sequence GGSLSR(SEQ ID NO: 62).
 12. The multispecific binder of claim 11, wherein theISVD that binds human serum albumin comprises the amino acid sequence(SEQ ID NO: 59) EVQLVESGGG VVQPGNSLRL SCAASGFTFS SFGMSWVRQAPGKGLEWVSS ISGSGSDTLYADSVKGRFTI SRDNAKTTLY LQMNSLRPED TALYYCTIGG SLSRSSQGTL VTVSSA.


13. An injection device or vessel that comprises the multispecificbinder of claim
 6. 14. A composition comprising a multispecific binderof claim 6 and at least one pharmaceutically acceptable carrier, diluentor excipient.
 15. The composition of claim 14, wherein the multispecificbinder comprises the amino acid sequence set forth in SEQ ID NO: 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, or
 124. 16. The composition of claim 15, wherein themultispecific binder comprises the amino acid sequence set forth in SEQID NO:110.
 17. A multispecific binder consisting of the amino acidsequence set forth in SEQ ID NO: 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or
 124. 18. Acomposition comprising the multispecific binder of claim 17 and at leastone pharmaceutically acceptable carrier, diluent or excipient.
 19. Amultispecific binder consisting of the amino acid sequence set forth inSEQ ID NO:
 110. 20. A composition comprising the multispecific binder ofclaim 19 and at least one pharmaceutically acceptable carrier, diluentor excipient.
 21. A multispecific binder comprising a programmed celldeath protein 1 (PD1) binder having the amino acid sequence set forth inSEQ ID NO: 57 and a lymphocyte activation gene 3 (LAG3) binder havingthe amino acid sequence set forth in SEQ ID NO:
 64. 22. Themultispecific binder of claim 21, wherein the multispecific binderfurther comprises a human serum albumin (HSA) binder having the aminoacid sequence set forth in SEQ ID NO:
 142. 23. A multispecific bindercomprising an N-terminal immunoglobulin single variable domain (ISVD)that binds programmed cell death protein 1 (PD1) comprising the aminoacid sequence set forth in SEQ ID NO: 57; an ISVD that binds lymphocyteactivation gene 3 (LAG3) comprising the amino acid sequence set forth inSEQ ID NO: 64; an ISVD that binds human serum albumin (HSA) comprisingthe amino acid sequence set forth in SEQ ID NO: 142; and a C-terminalAlanine.
 24. A multispecific binder comprising an N-terminalimmunoglobulin single variable domain (ISVD) that binds programmed celldeath protein 1 (PD1) comprising the amino acid sequence set forth inSEQ ID NO: 57; a 35GS linker comprising the amino acid sequence setforth in SEQ ID NO: 58; an ISVD that binds lymphocyte activation gene 3(LAG3) comprising the amino acid sequence set forth in SEQ ID NO: 64; a35GS linker comprising the amino acid sequence set forth in SEQ ID NO:58; an ISVD that binds human serum albumin (HSA) comprising the aminoacid sequence set forth in SEQ ID NO: 142; and a C-terminal Alanine. 25.A composition comprising the multispecific binder of claim 21 and atleast one pharmaceutically acceptable carrier, diluent or excipient. 26.A multispecific binder comprising the amino acid sequence set forth inSEQ ID NO:
 110. 27. A composition comprising the multispecific binder ofclaim 26 and at least one pharmaceutically acceptable carrier, diluentor excipient.
 28. A composition comprising the multispecific binder ofclaim 24 and at least one pharmaceutically acceptable carrier, diluentor excipient.
 29. The injection device or vessel of claim 13, whereinthe multispecific binder is in association with a further therapeuticagent.
 30. A composition comprising (a) a multispecific bindercomprising an N- terminal immunoglobulin single variable domain (ISVD)that binds programmed cell death protein 1 (PD1) comprising the aminoacid sequence set forth in SEQ ID NO: 57; an ISVD that binds lymphocyteactivation gene 3 (LAG3) comprising the amino acid sequence set forth inSEQ ID NO: 64; an ISVD that binds human serum albumin (HSA) comprisingthe amino acid sequence set forth in SEQ ID NO: 142; and a C-terminalAlanine, and, (b) at least one pharmaceutically pharmaceuticallyacceptable carrier, diluent or excipient.
 31. A composition comprising(a) a multispecific binder comprising an N-terminal immunoglobulinsingle variable domain (ISVD) that binds programmed cell death protein 1(PD1) comprising the amino acid sequence set forth in SEQ ID NO: 57; a35GS linker comprising the amino acid sequence set forth in SEQ ID NO:58; an ISVD that binds lymphocyte activation gene 3 (LAG3) comprisingthe amino acid sequence set forth in SEQ ID NO: 64; a 35GS linkercomprising the amino acid sequence set forth in SEQ ID NO: 58; an ISVDthat binds human serum albumin (HSA) comprising the amino acid sequenceset forth in SEQ ID NO: 142; and a C-terminal Alanine, and, (b) at leastone pharmaceutically pharmaceutically acceptable carrier, diluent orexcipient.